Nucleic acids of the human ABCA5, ABCA6, ABCA9, AND ABCA10 Genes, vectors containing such nucleic acids, and uses thereof

ABSTRACT

The present invention relates to nucleic acids corresponding to various exons of ABCA5, ABCA6, ABCA9, and ABCA10 genes as well as cDNAs encoding the novel full length of ABCA5, ABCA6, ABCA9, and ABCA10 proteins. The invention also relates to means for the detection of polymorphisms in general, and of mutations in particular, in the ABCA5, ABCA6, ABCA9, and ABCA10 genes or in the corresponding protein produced by the allelic form of the ABCA5, ABCA6, ABCA9, and ABCA10 genes.

[0001] Under the provisions of Section 119 of 35 U.S.C., his application claims priority to French application No. 00403440.1, filed Dec. 7, 2000. This application also claims the benefit of U.S. Provisional Application No. 60/263,231, filed Jan. 23, 2001, which is incorporated herein in its entirety.

[0002] The present invention relates to novel nucleic acids corresponding to the ABCA5, ABCA6, ABCA9, and ABCA10 genes, and cDNAs encoding novel ABCA5, ABCA6, ABCA9, and ABCA10 proteins. The invention also relates to means for the detection of polymorphisms in general, and mutations in particular in the ABCA5, ABCA6, ABCA9, and ABCA10 genes or corresponding proteins produced by the allelic forms of the ABCA5, ABCA6, ABCA9, and ABCA10 genes.

[0003] The genes of the ABC (ATP-binding cassette transporter) superfamily encode active transporter proteins, which are extremely well conserved during evolution, from bacteria to humans (Ames and Lecar, FASEB J., 1992, 6, 2660-2666). The ABC proteins are involved in extra- and intracellular membrane transport of various substrates, for example, ions, amino acids, peptides, sugars, vitamins, and steroid hormones. Among the 40 characterized humans members of the ABC superfamily, 11 members have been described as associated with human disease, such as, inter alia, ABCA1, ABCA4 (ABCR), and ABCC7 (CFTR), which are thought to be involved in Tangier disease (Bodzioch M et al., Nat. Genet., 1999, 22(4); 347-351; Brooks-Wilson et al., Nat Genet,1999, 22(4), 336-345; Rust S et al., Nat. Genet., 1999, 22, 352-355; Remaley A T et al., ), Stargardt disease (Lewis R A et al., Am. J. Hum. Genet., 1999, 64, 422-434), and cystic fibrosis (Riordan J M et al., Science, 1989, 245, 1066-1073), respectively. These associations reveal the importance of ABC gene family function. The discovery of new family gene members should provide insights into the physiopathology of additional human diseases.

[0004] The prototype ABC protein binds ATP and uses the energy from ATP hydrolysis to drive the transport of various molecules across cell membranes. The prototype protein contains two ATP-binding domains (nucleotide binding fold, NBF) and two transmembrane (TM) domains. The genes are typically organized as full transporters containing two of each domain, or half transporters with only one of each domain. Most full transporters are arranged in a TM-NBF-TM-NBF fashion (Dean et al., Curr Opin Genet, 1995, 5, 79-785).

[0005] Analysis of amino acids sequence alignments of the ATP-binding domains has allowed the ABC genes to be separated into sub-families (Allikmets et al., Hum Mol Genet, 1996, 5, 1649-1655). Currently, according to the recent HUGO classification, seven ABC gene sub-families named ABC (A to G) have been described in the human genome (ABC1, CFTR/MRP, MDR, ABC8, ALD, GCN20, OABP) with all except one (OABP) containing multiple members. For the most part, these sub-families contain genes that also display considerable conservation in the transmembrane domain sequences and have similar gene organization. However, ABC proteins transport very varied substrates, and some members of different sub-families have been shown to share more similarity in substrate recognition than do proteins within the same sub-family. Five of the sub-families also are represented in the yeast genome, indicating that these groups have been retained from an early time in the evolution of eukaryotes (Decottignies et al., Nat Genet, 1997, 137-45; Michaelis et al., 1995, Cold Spring Harbor Laboratory Press).

[0006] Several ABC transport proteins that have been identified in humans are associated with diseases. For example, cystic fibrosis is caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene (Riordan J M et al., Science, 1989, 245,1066-1073). Moreover, some multiple drug resistance phenotypes in tumor cells have been associated with the gene encoding the MDR (multi-drug resistance) protein, which also has an ABC transporter structure (Anticancer Drug Des. 1999 Apr14(2):115-31.). Other ABC transporters have been associated with neuronal and tumor conditions (U.S. Pat. No. 5,858,719) or potentially involved in diseases caused by impairment of the homeostasis of metals (Biochim. Biophys. Acta. 1999 Dec 6;1461(2):18-404. ). Likewise, another ABC transporter, designated PFIC2, appears to be involved in a progressive familial intrahepatic cholestasia form, this protein potentially being responsible, in humans, for the export of bile salts (Strautnieks S S et al, Nat Genet, 1998, 20, 233-238).

[0007] Among the ABC sub-families, the ABCA gene subfamily is probably the most evolutionarily complex. The ABCA genes and OABP are the only two sub-families of ABC genes that do not have identifiable orthologs in the yeast genome (Decottignies and Goffeau, 1997; Michaelis and Berkower, 1995). There is, however, at least one ABCA-related gene in C. elegans (ced-7) and several in Drosophila. Thus the ABCA genes appear to have diverged after eukaryotes became multicellular and developed more sophisticated transport requirements. To date, eleven members of the human ABCA sub-family have been described, making it the largest such group.

[0008] Full sequences of four genes of the ABCA sub-family have been described, revealing a complex exon-intron structure. The best characterized ABCA genes are ABCA4, and ABCA1. In mammals, the ABCA1 gene is highly expressed in macrophages and monocytes and is associated with the engulfment of apoptotic cells (Luciani et al, Genomics (1994) 21,150-9; Moynault et al., Biochem Soc Trans (1998) 26, 629-35; Wu et al., Cell (1998) 93, 951-60). The ced-7 gene, the ortholog of ABCA1 in C. elegans, also plays a role in the recognition and engulfment of apoptotic cells, suggesting a conserved function. Recently ABCA1 was demonstrated to be the gene responsible for Tangier disease, a disorder characterized by high levels of cholesterol in peripheral tissues and a very low level of HDLs, and for familial hypoalphalipoproteinemia (FHD) (Bodzioch et al., Nat Genet (1999) 22, 347-51; Brooks-Wilson et al., Nat Genet (1999) 336-45; Rust et al., Nat Genet (1999) 22, 352-5; Marcil et al., The Lancet (1999) 354,1341-46). The ABCA1 protein is proposed to function in the reverse transport of cholesterol from peripheral tissues via an interaction with the apolipoprotein 1 (ApoA-1) of HDL (Wang et al., J. Biol. Chem. (2000)).

[0009] The ABCA2 gene is highly expressed in the brain and ABCA3 in the lung, but no function has been ascribed to these loci. The ABCA4 gene is exclusively expressed in the rod photoreceptors of the retina, and mutations thereof are responsible for several pathologies of human eyes, such as retinal degenerative disorders (Allikmets et al., Science (1997) 277, 1805-1807; Allikmets et al., Nat Genet (1997) 15, 236-246; Sun et al., J Biol Chem (1999) 8269-81; Weng et al., Cell (1999) 98, 13-23; Cremers et al., Hum Mol Genet (1998) 7, 355-362; Martinez-Mir et al., Genomics (1997) 40, 142-146). ABCA4 is believed to transport retinal and/or retinal-phospholipid complexes from the rod photoreceptor outer segment disks to the cytoplasm, thereby facilitating phototransduction.

[0010] Characterization of new genes from the ABCA subfamily is likely to yield biologically important transporters that may have an translocase activity for membrane lipid transport and may play a major role in human pathologies.

[0011] Lipids are water-insoluble organic biomolecules, which are essential components with diverse biological functions, including the storage, transport, and metabolism of energy, and membrane structure and fluidity. Lipids are derived from two sources in humans and other animals: some lipids are ingested as dietary fats and oils and other lipids are biosynthesized by the human or animal. In mammals, at least 10% of the body weight is lipid, the bulk of which is in the form of triacylglycerols.

[0012] Triacylglycerols, also known as triglycerides and triacylglycerides, are made up of three fatty acids esterified to glycerol. Dietary triacylglycerols are stored in adipose tissues as a source of energy or hydrolyzed in the digestive tract by triacylglycerols lipases, the most important of which is pancreatic lipase. Triacylglycerols are transported between tissues in the form of lipoproteins.

[0013] Lipoproteins are micelle-like assemblies found in plasma and contain varying proportions of different types of lipids and proteins (called apoproteins). There are five main classes of plasma lipoproteins, the major function of which is lipid transport. These classes are, in order of increasing density, chylomicrons, very low density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low density lipoproteins (LDL), and high density lipoproteins (HDL). Although many types of lipids are found associated with each lipoprotein class, each class transports predominantly one type of lipid: triacylglycerols are transported in chylomicrons, VLDL, and IDL, while phospholipids and cholesterol esters are transported in HDL and LDL, respectively.

[0014] Phospholipids are di-fatty acid esters of glycerol phosphate, also containing a polar group coupled to the phosphate. Phospholipids are important structural components of cellular membranes. Phospholipids are hydrolyzed by enzymes called phospholipases. Phosphatidylcholine, an exemplary phospholipid, is a major component of most eukaryotic cell membranes.

[0015] Cholesterol is the metabolic precursor of steroid hormones and bile acids, as well as an essential constituent of cell membranes. In humans and other animals, cholesterol is ingested in the diet and also synthesized by the liver and other tissues. Cholesterol is transported between tissues in the form of cholesteryl esters in LDLs and other lipoproteins.

[0016] Membranes surround every living cell and serve as a barrier between the intracellular and extracellular compartments. Membranes also enclose the eukaryotic nucleus, make up the endoplasmic reticulum, and serve specialized functions such as in the myelin sheath that surrounds axons. A typical membrane contains about 40% lipid and 60% protein, but there is considerable variation. The major lipid components are phospholipids, specifically phosphatidylcholine and phosphatidylethanolamine, and cholesterol. The physicochemical properties of membranes, such as fluidity, can be changed by modification of either the fatty acid profiles of the phospholipids or the cholesterol content. Modulating the composition and organization of membrane lipids also modulates membrane-dependent cellular functions, such as receptor activity, endocytosis, and cholesterol flux.

[0017] High-density lipoproteins (HDL) are one of the five major classes of lipoproteins circulating in blood plasma. These lipoproteins are involved in various metabolic pathways such as lipid transport, the formation of bile acids, steroidogenesis, cell proliferation, and, in addition, interfere with the plasma proteinase systems.

[0018] HDLs are perfect free cholesterol acceptors and, in combination with enzymatic activities such as that of the cholesterol ester transfer protein (CETP), the lipoprotein lipase (LPL), the hepatic lipase (HL), and the lecithin:cholesterol acyltransferase (LCAT), play a major role in the reverse transport of cholesterol, i.e., the transport of excess cholesterol in peripheral cells to the liver for its elimination from the body in the form of bile acid. It has been demonstrated that the HDLs play a central role in the transport of cholesterol from peripheral tissues to the liver.

[0019] Various diseases linked to HDL deficiency have been described, including Tangiers disease, FHD disease, and LCAT deficiency. In addition, HDL-cholesterol deficiencies have been observed in patients suffering from malaria and diabetes (Kittl et al., 1992. Wein Klin Wochenschr 104 :21-4; Nilsson et al., 1990, J. Intern. Med., 227:151-5; Djoumessi, 1989, Pathol Biol., 37:909-11; Mohanty et al., 1992. Ann Trop Med Parasitol., 86 :601-6; Maurois et al., 1985, Biochimie, 67 :227-39; Grellier et al., 1997. Vox Sang. 72 :211-20; Agbedana et al., 1990, Ann Trop Med Parasitol., 84 :529-30; Erel et al., 1998, Haematologia, Budap, 29 :207-12; Cuisinier et al., 1990, Med Trop, 50 :91-5; Chander et al., 1998, Indian J Exp Biol., 36 :371-4; Efthimiou et al., 1992, Wein Klin Wochenschr., 104 :705-6; Baptista et al., 1996. Parasite, 3:335-40; Davis et al., 1993, J. Infect. 26 :279-85; Davis et al., 1995, J. Infect. 31:181-8; Pirich et al., 1993, Semin Thromb Hemost., 19:138-43; Tomlinson and Raper, 1996, Nat. Biotechnol., 14:717-21; Hager and Hajduk, 1997, Nature 385:823-6; Kwiterovich, 1995, Ann NY Acad Sci., 748 :313-30 ; Syvanne et al. 1995, Circulation, 92:364-70; and Syvanne et al., 1995, J. Lipid Res., 36:573-82). The deficiency involved in Tangier and/or FHD disease is linked to a cellular defect in the translocation of cellular cholesterol that causes a degradation of HDLs and leads to a disruption in lipoprotein metabolism.

[0020] Atherosclerosis is defined in histological terms by deposits (lipid or fibrolipid plaques) of lipids and of other blood derivatives in blood vessel walls, especially the large arteries (aorta, coronary arteries, carotid). These plaques, which are more or less calcified according to the degree of progression of the atherosclerosis process, may be coupled with lesions and are associated with the accumulation in the vessels of fatty deposits consisting essentially of cholesterol esters. Development of these plaques is accompanied by a thickening of the vessel wall, hypertrophy of the smooth muscle, appearance of foam cells (lipid-laden cells resulting from uncontrolled uptake of cholesterol by recruited macrophages), and accumulation of fibrous tissue. The atheromatous plaque protrudes markedly from the wall, endowing it with a stenosing character responsible for vascular occlusions by atheroma, thrombosis, or embolism, which occur in those patients who are most severely affected. These lesions can lead to serious cardiovascular pathologies, such as myocardial infarction, sudden death, cardiac insufficiency, and stroke.

[0021] Mutations within genes that play a role in lipoprotein metabolism have been identified. Specifically, several mutations in the apolipoprotein apoA-I gene have been characterized. These mutations are rare and may lead to a lack of production of apoA-I. Mutations in the genes encoding LPL or its activator apoC-II are associated with severe hypertriglyceridemias and substantially reduced HDL-C levels. Mutations in the gene encoding the enzyme LCAT also are associated with severe HDL deficiency.

[0022] In addition, dysfunctions in the reverse transport of cholesterol may be induced by physiological deficiencies affecting one or more of the steps in the transport of stored cholesterol, from the intracellular vesicles to the membrane surface where it is accepted by the HDLs.

[0023] Therefore, a need exists to identify genes involved in any of the steps in the metabolism of cholesterol and/or lipoproteins, and, in particular, genes associated with dysfunctions in the reverse transport of cholesterol from peripheral cells to the liver.

[0024] Applicants have discovered and characterized a gene cluster containing 4 new genes belonging to the ABCA protein sub-family, which have been designated ABCA5, ABCA6, ABCA9, and ABCA10. These new genes appear to be closely related to other ABCA subfamily members such as ABCA1 and ABCA8, particularly in the ATP-binding domain and in the C-terminal ATP binding domains. The newly discovered genes also show considerable conservation of amino acid sequence, particularly within the transmembrane region (TM) and the ATP-binding regions (NBD), and have a similar gene organization.

[0025] Surprisingly, Applicants have found these genes to be organized in a single large cluster on chromosome 17q24, in a head-to-tail fashion, with a similar intron/exon organization, suggesting that they have arisen from tandem duplication and that they may form a distinct functional group with the ABCA subfamily.

[0026] Furthermore, each of the newly discovered genes is transcribed with a tissue-specific distribution and presents a heterogenous pattern of expression, suggesting a regional and probably functional specialization of the corresponding proteins.

SUMMARY OF THE INVENTION

[0027] The present invention relates to nucleic acids corresponding to the various human ABCA5, ABCA6, ABCA9, and ABCA10 genes, which are likely to be involved in the reverse transport of cholesterol, as well as in the membrane transport of lipophilic molecules, in particular, inflammation-mediating substances such as prostaglandins and prostacyclins, or in any pathology whose candidate chromosomal region is situated on chromosome 17, more precisely on the 17q arm and, still more precisely, in the 17q24 locus.

[0028] Thus, a first subject of the invention is a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOs: 1-4, and 9-126, or a complementary nucleotide sequence thereof.

[0029] The invention also relates to a nucleic acid comprising at least 8 consecutive nucleotides of a nucleotide sequence of any one of SEQ ID NOs: 1-4, and 9-126 or a complementary nucleotide sequence thereof.

[0030] The invention also relates to a nucleic acid having at least 80% nucleotide identity with a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOs: 1-4, and 9-126 or a complementary nucleotide sequence thereof.

[0031] The invention also relates to a nucleic acid having at least 85%, at least 90%, at least 95%, or at least 98% nucleotide identity with a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence thereof.

[0032] The invention also relates to a nucleic acid hybridizing, under high stringency conditions, with a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence thereof.

[0033] The invention also relates to nucleic acids, particularly cDNA molecules, which encode the full length human ABCA5, ABCA6, ABCA9, or ABCA10 proteins. Thus, the invention relates to a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOS: 1-4 or of a complementary nucleotide sequence.

[0034] The invention also relates to a nucleic acid comprising a nucleotide sequence as depicted in SEQ ID NOS: 1-4 or a complementary nucleotide sequence.

[0035] According to the invention, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 1 encodes a full length ABCA5 polypeptide of 1642 amino acids comprising the amino acid sequence of SEQ ID NO: 5.

[0036] According to the invention, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 2 encodes a full length ABCA6 polypeptide of 1617 amino acids comprising the amino acid sequence of SEQ ID NO: 6.

[0037] According to the invention, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 3 encodes a full length ABCA9 polypeptide of 1624 amino acids comprising the amino acid sequence of SEQ ID NO: 7.

[0038] According to the invention, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 4 encodes a full length ABCA10 polypeptide of 1543 amino acids comprising the amino acid sequence of SEQ ID NO: 8.

[0039] Thus, the invention also relates to a nucleic acid encoding a polypeptide comprising an amino acid sequence of any one of SEQ ID NOS: 5-8.

[0040] Thus, the invention also relates to a polypeptide comprising an amino acid sequence of any one of SEQ ID NOS: 5-8.

[0041] The invention also relates to a polypeptide comprising an amino acid sequence as depicted in any one of SEQ ID NOS: 5-8.

[0042] The invention also relates to a means for detecting polymorphisms in general, and mutations in particular, in the ABCA5, ABCA6, ABCA9, and ABCA10 genes or in the corresponding proteins produced by the allelic form of these genes.

[0043] According to another aspect, the invention relates to the nucleotide sequences of the ABCA5, ABCA6, ABCA9, and ABCA10 genes comprising at least one biallelic polymorphism such as, for example, a substitution, addition, or deletion of one or more nucleotides.

[0044] The invention also encompasses nucleotide probes and primers hybridizing with a nucleic acid sequence located in the region of any one of the ABCA5, ABCA6, ABCA9, and ABCA10 nucleic acids (genomic DNA, messenger RNA, cDNA), in particular, a nucleic acid sequence comprising any one of the mutations or polymorphisms.

[0045] The nucleotide probes or primers according to the invention comprise at least 8 consecutive nucleotides of a nucleic acid comprising any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence thereof. Nucleotide probes or primers according to the invention may have a length of 10, 12, 15, 18 or 20 to 25, 35, 40, 50, 70, 80, 100, 200, 500, 1000, 1500 consecutive nucleotides of a nucleic acid according to the invention, for example, a nucleic acid comprising any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence thereof.

[0046] Alternatively, a nucleotide probe or primer according to the invention will consist of and/or comprise fragments having a length of 12, 15, 18, 20, 25, 35, 40, 50, 100, 200, 500, 1000, 1500 consecutive nucleotides of a nucleic acid according to the invention, for example, a nucleic acid comprising any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence thereof.

[0047] The definition of a nucleotide probe or primer according to the invention, therefore, encompasses oligonucleotides that hybridize, under high stringency hybridization conditions defined below, with a nucleic acid comprising any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence thereof.

[0048] The probes and primers according to the invention may also comprise all or part of a nucleotide sequence comprising any one of SEQ ID NOs: 127-217 or a complementary nucleotide sequence thereof.

[0049] Nucleotide primers according to the invention may be used to amplify any one of the nucleic acids according to the invention, for example, a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence thereof.

[0050] According to the invention, some nucleotide primers specific for an ABCA5 gene, may be used to amplify a nucleic acid comprising SEQ ID NO: 1 and comprise a nucleotide sequence of any one of SEQ ID NOs: 127-150, or a complementary nucleotide sequence thereof.

[0051] The invention also relates to nucleotide primers that are specific for an ABCA6 gene, which may be used to amplify a nucleic acid comprising any one of SEQ ID NOs: 2 and 9-47 and comprise a nucleotide sequence of any one of SEQ ID NOs: 151-177 or a complementary nucleotide sequence.

[0052] The invention is further directed to nucleotide primers specific for an ABCA9 gene, which may be used to amplify a nucleic acid comprising any one of SEQ ID NOs: 3, and 48-86 and comprise a nucleotide sequence of any one of SEQ ID NOs: 178-209 or a complementary nucleotide sequence.

[0053] The present invention is further directed to nucleotide primers specific for an ABCA10 gene, which may be used to amplify a nucleic acid comprising any one of SEQ ID NOs: 4, and 87-126 and comprise a nucleotide sequence of any one of SEQ ID NOs: 210-217 or a complementary nucleotide sequence.

[0054] Another subject of the invention relates to a method of amplifying a nucleic acid according to the invention, for example, a nucleic acid comprising a) any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence thereof, or b) as depicted in any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence thereof, contained in a sample, said method comprising:

[0055] a) bringing the sample in which the presence of the target nucleic acid is suspected into contact with a pair of nucleotide primers whose hybridization position is located, respectively, on the 5′ side and on the 3′ side of the region of the target nucleic acid whose amplification is sought, in the presence of the reagents necessary for the amplification reaction;

[0056] b) performing an amplification reaction; and, optionally,

[0057] c) detecting the amplified nucleic acids.

[0058] The present invention also relates to a method of detecting the presence of a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126, or a complementary nucleotide sequence, or a nucleic acid fragment or variant of any one of SEQ ID NOs: 1-4 and 9-126 , or a complementary nucleotide sequence in a sample, said method comprising:

[0059] 1) bringing one or more nucleotide probes according to the invention into contact with the sample to be tested; and

[0060] 2) detecting the complex that may have formed between the probe(s) and the nucleic acid present in the sample.

[0061] According to an embodiment of the method of detection according to the invention, the oligonucleotide probes are immobilized on a support.

[0062] According to another embodiment, the oligonucleotide probes comprise a detectable marker.

[0063] Another subject of the invention is a box or kit for amplifying all or part of a nucleic acid comprising a) any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence thereof or b) any of the sequences as depicted in any one of SEQ ID NOs: 1-4 and 9-126 or of a complementary nucleotide sequence thereof, said box or kit comprising:

[0064] 1) a pair of nucleotide primers in accordance with the invention, whose hybridization position is located, respectively, on the 5′ side and on the 3′ side of the target nucleic acid whose amplification is sought; and, optionally,

[0065] 2) reagents necessary for an amplification reaction.

[0066] Such an amplification box or kit will preferably comprise at least one pair of nucleotide primers as described above.

[0067] The invention also relates to a box or kit for detecting the presence of a nucleic acid according to the invention in a sample, said box or kit comprising:

[0068] a) one or more nucleotide probes according to the invention;

[0069] b) appropriate reagents necessary for a hybridisation reaction.

[0070] According to one aspect, the detection box or kit is characterized in that the nucleotide probe(s) and primer(s)are immobilized on a support.

[0071] According to another aspect, the detection box or kit is characterized in that the nucleotide probe(s) and primer(s) comprise a detectable marker.

[0072] According to an embodiment of the detection kit described above, such a kit will comprise a plurality of oligonucleotide probes and/or primers in accordance with the invention that may be used to detect target nucleic acids of interest or, alternatively, to detect mutations in the coding and/or the non-coding regions of the nucleic acids according to the invention. According to another embodiment of the invention, the target nucleic acid comprises a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126 or of a complementary nucleic acid sequence. Alternatively, the target nucleic acid is a nucleic acid fragment or variant of a nucleic acid comprising any one of SEQ ID NOs: 1-4 and 9-126 or of a complementary nucleotide sequence.

[0073] According to another embodiment, a primer according to the invention comprises all or part of any one of SEQ ID NOs: 1-4, and 9-217 or a complementary sequence.

[0074] The invention also relates to a recombinant vector comprising a nucleic acid according to the invention. Such a recombinant vector may comprise a nucleic acid selected from:

[0075] a) a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence thereof,

[0076] b) a nucleic acid comprising a nucleotide sequence as depicted in any one of SEQ ID NOs: 1-4 and 9-126,or a complementary nucleotide sequence thereof,

[0077] c) a nucleic acid having at least eight consecutive nucleotides of a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence thereof;

[0078] d) a nucleic acid having at least 80% nucleotide identity with a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence thereof;

[0079] e) a nucleic acid having at least 85%, at least 90%, at least 95%, or at least 98% nucleotide identity with a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence thereof;

[0080] f) a nucleic acid hybridizing, under high stringency hybridization conditions, with a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence; and

[0081] g) a nucleic acid encoding a polypeptide comprising an amino acid sequence of any one of SEQ ID NOS: 5-8.

[0082] According to one embodiment, a recombinant vector according to the invention is used to amplify a nucleic acid inserted therein, following transformation or transfection of a desired cellular host.

[0083] According to another embodiment, a recombinant vector according to the invention is an expression vector comprising, in addition to a nucleic acid in accordance with the invention, a regulatory signal or nucleotide sequence that directs or controls transcription and/or translation of the nucleic acid and its encoded mRNA.

[0084] According to yet another embodiment, a recombinant vector according to the invention may comprise, for example, the following components:

[0085] (1) an element or signal for regulating the expression of the nucleic acid to be inserted, such as a promoter and/or enhancer sequence;

[0086] (2) a nucleotide coding region comprised within a nucleic acid according to the invention to be inserted into such a vector, said coding region being placed in phase with the regulatory element or signal described in (1); and

[0087] (3) an appropriate nucleic acid for initiation and termination of transcription of the nucleotide coding region of the nucleic acid described in (2).

[0088] The present invention also relates to a defective recombinant virus comprising a cDNA encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides involved in the transport of lipophilic substances, for example, mediators of inflammation, or in any pathology whose candidate chromosomal region is situated on chromosome 17, more precisely on the 17q arm, and, still more precisely, in the 17q24 locus.

[0089] In another embodiment of the invention, the defective recombinant virus comprises a genomic DNA (gDNA) encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides involved in the transport of lipophilic substances, inflammatory lipophilic substances. The ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides may comprise amino acid sequences selected from SEQ ID NOS: 5-8, respectively.

[0090] In another embodiment, the invention relates to a defective recombinant virus comprising a nucleic acid encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides involved in the transport of inflammatory lipophilic substances, under the control of a promoter chosen from (Rous sarcoma virus (RSV)-LTR or the cytomegalovirus (CMV) early promoter.

[0091] According to an embodiment of the invention, a method of introducing a nucleic acid according to the invention into a host cell, for example, a host cell obtained from a mammal, in vivo, comprises a step during which a preparation comprising a pharmaceutically compatible vector and a “naked” nucleic acid according to the invention, placed under the control of appropriate regulatory sequences, is introduced by local injection at the site of the chosen tissue, for example, a smooth muscle tissue, the “naked” nucleic acid being absorbed by the cells of this tissue.

[0092] According to another embodiment of the invention, a composition is provided for the in vivo production of any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins. This composition comprises a nucleic acid encoding the ABCA5, ABCA6, ABCA9, or ABCA10 polypeptide placed under the control of appropriate regulatory sequences in solution in a physiologically-acceptable vehicle and/or excipient.

[0093] Therefore, the present invention also relates to a composition comprising a nucleic acid encoding any one of the ABCA5, ABCA6, ABCA9, ABCA10 polypeptides, wherein the polypeptide comprises an amino acid sequence selected from SEQ ID NOS: 5-8, and wherein the nucleic acid is placed under the control of appropriate regulatory elements.

[0094] Consequently, the invention also relates to a pharmaceutical composition intended for the prevention of or treatment of a patient or subject affected by a dysfunction in the reverse transport of cholesterol or in the transport of inflammatory lipophilic substances, wherein the composition comprises a nucleic acid encoding any one of ABCA5, ABCA6, ABCA9, and ABCA10 proteins, in combination with one or more physiologically compatible excipients.

[0095] Such a composition may comprise a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126, wherein the nucleic acid is placed under the control of an appropriate regulatory element or signal.

[0096] In addition, the present invention is directed to a pharmaceutical composition intended for the prevention of or treatment of a patient or a subject affected by a dysfunction in the reverse transport of cholesterol or in the transport of liphophilic substances mediating inflammation, comprising a recombinant vector according to the invention in combination with one or more physiologically-compatible excipients.

[0097] The invention also relates to the use of a nucleic acid according to the invention encoding any one of ABCA5, ABCA6, ABCA9, and ABCA10 proteins for the manufacture of a medicament intended for the prevention of atherosclerosis, for example, for the treatment of subjects affected by a dysfunction of cholesterol reverse transport or transport of liphophilic substances mediating inflammation.

[0098] The invention also relates to the use of a recombinant vector according to the invention comprising a nucleic acid encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins for the manufacture of a medicament intended for the prevention of atherosclerosis in various forms or, for example, for the treatment of subjects affected by a dysfunction of cholesterol reverse transport or transport of liphophilic substances mediating inflammation.

[0099] The subject of the invention is therefore also a recombinant vector comprising a nucleic acid according to the invention that encodes any one of the ABCA5, ABCA6, ABCA9 and ABCA10 proteins or polypeptides involved in the metabolism of cholesterol or transport of liphophilic substances mediating inflammation.

[0100] The invention also relates to the use of such a recombinant vector for the preparation of a pharmaceutical composition intended for the treatment and/or for the prevention of diseases or conditions associated with deficiency of lipophilic substances signaling inflammation, or deficiency of the reverse transport of cholesterol, or deficiency of the transport of inflammatory lipophilic substances.

[0101] The present invention also relates to the use of cells genetically modified ex vivo with a recombinant vector according to the invention or to cells producing a recombinant vector, wherein the cells may be implanted in the body, to allow a prolonged and effective expression in vivo of any one of biologically active ABCA5, ABCA6, ABCA9, or ABCA10 polypeptide.

[0102] The invention also relates to the use of a nucleic acid according to the invention encoding any one of the ABCA5, ABCA6, ABCA9 and ABCA10 proteins for the manufacture of a medicament intended for the prevention and/or the treatment of subjects affected by a dysfunction of cholesterol reverse transport or inflammatory lipophilic substances transport.

[0103] The invention also relates to the use of a recombinant vector according to the invention comprising a nucleic acid encoding any one of the ABCA5, ABCA6, ABCA9 and ABCA10 polypeptides according to the invention for the manufacture of a medicament intended for the prevention and/or the treatment of subjects affected by a dysfunction of the reverse transport of cholesterol or inflammatory lipophilic substances transport.

[0104] The invention also relates to the use of a recombinant host cell according to the invention, comprising a nucleic acid encoding any one of the ABCA5, ABCA6, ABCA9 and ABCA10 polypeptides according to the invention for the manufacture of a medicament intended for the prevention and/or the treatment of subjects affected by a dysfunction of cholesterol reverse transport.

[0105] The invention also relates to the use of a recombinant vector according to the invention, for example, a defective recombinant virus, for the preparation of a pharmaceutical composition for the treatment and/or prevention of pathologies linked to the dysfunction of cholesterol reverse transport or inflammatory lipophilic substances transport.

[0106] The invention relates to the use of such a recombinant vector or defective recombinant virus for the preparation of a pharmaceutical composition intended for the treatment and/or for the prevention of cardiovascular disease linked to a deficiency in the reverse transport of cholesterol. Thus, the present invention also relates to a pharmaceutical composition comprising one or more recombinant vectors or defective recombinant viruses according to the invention.

[0107] The present invention also relates to the use of cells genetically modified ex vivo with a virus according to the invention and to cells producing such viruses, which may be implanted in the body, allowing a prolonged and effective expression in vivo of any one of biologically active of ABCA5, ABCA6, ABCA9 or ABCA10 protein.

[0108] The present invention shows that it is possible to incorporate a nucleic acid encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides according to the invention into a viral vector, and that these vectors make it possible to express a biologically active, mature polypeptide. Moreover, the invention shows that the in vivo expression of any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins may be obtained by direct administration of an adenovirus or by implantation of a producing cell or of a cell genetically modified by an adenovirus or by a retrovirus incorporating such a nucleic acid.

[0109] In this regard, another subject of the invention is any mammalian cell infected with one or more defective recombinant viruses according to the invention. The invention also encompases any population of human cells infected with these viruses. These may be, for example, of blood origin (totipotent stem cells or precursors), fibroblasts, myoblasts, hepatocytes, keratinocytes, smooth muscle, endothelial cells, glial cells, and the like.

[0110] Another subject of the invention is an implant comprising mammalian cells infected with one or more defective recombinant viruses according to the invention or cells producing recombinant viruses and an extracellular matrix. In general, the implants according to the invention comprise 10⁵ to 10¹⁰ cells. In one embodiment, the implants comprise 10⁶ to 10⁸ cells.

[0111] In the implants of the invention, the extracellular matrix may additionally comprise a gelling compound and, optionally, a support for the anchorage of the cells.

[0112] The invention also relates to a recombinant host cell comprising a nucleic acid of the invention, for example, a nucleic acid comprising any one of SEQ ID NOS: 1-4 and 9-126 or of a complementary nucleotide sequence.

[0113] The invention also relates to a recombinant host cell comprising a nucleic acid of the invention, for example, a nucleic acid comprising a nucleotide sequence as depicted in any one SEQ ID NOS: 1-4 and 9-126 or of a complementary nucleotide sequence.

[0114] According to another aspect, the invention encompasses a recombinant host cell comprising a recombinant vector according to the invention. Therefore, the invention also relates to a recombinant host cell comprising a recombinant vector comprising any of the nucleic acids of the invention, for example, a nucleic acid comprising any one nucleotide sequence of SEQ ID NOS: 1-4 and 9-126 or a complementary nucleotide sequence thereof.

[0115] The invention also relates to a recombinant host cell comprising a recombinant vector comprising a nucleic acid comprising any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence thereof.

[0116] The invention also relates to a recombinant host cell comprising a recombinant vector comprising a nucleic acid comprising a nucleotide sequence as depicted in any one of SEQ ID NOs:1-4 and 9-126 or of a complementary nucleotide sequence thereof.

[0117] The invention also relates to a recombinant host cell comprising a recombinant vector comprising a nucleic acid encoding a polypeptide comprising any one amino acid sequence of SEQ ID NOs: 5-8.

[0118] The invention also relates to a method for the production of a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 1-4 and 9-126 or of a peptide fragment or a variant thereof, said method comprising:

[0119] a) inserting a nucleic acid encoding said polypeptide into an appropriate vector;

[0120] b) culturing, in an appropriate culture medium under conditions that allow the expression of said polypeptide, a previously transformed host cell or transfecting a host cell with the recombinant vector of step a);

[0121] c) recovering the conditioned culture medium or lysing the host cell, for example, by sonication or by osmotic shock;

[0122] d) separating and purifying said polypeptide from said culture medium or, alternatively, from the cell lysates obtained in step c); and

[0123] e) where appropriate, characterizing the recombinant polypeptide produced.

[0124] A polypeptide termed “homologous” to a polypeptide having an amino acid sequence selected from SEQ ID NOS: 5-8 also is part of the invention. Such a homologous polypeptide comprises an amino acid sequence possessing one or more substitutions of an amino acid by an equivalent amino acid.

[0125] The ABCA5, ABCA6, ABCA9, ABCA10 polypeptides according to the invention, including 1) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-8, 2) a polypeptide fragment or variant of a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-8, and 3) a polypeptide termed “homologous” to a polypeptide comprising amino acid sequence selected from SEQ ID NOs: 5-8.

[0126] In one embodiment, an antibody according to the invention is directed against 1) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-8, 2) a polypeptide fragment or variant of a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 5-8, or 3) a polypeptide termed “homologous” to a polypeptide comprising amino acid sequence selected from SEQ ID NOS: 5-8. Such an antibody may be produced by any known techniques, including the trioma technique or the hybridoma technique described by Kozbor et al. (Immunology Today, (1983) 4:72).

[0127] Thus, the subject of the invention is, in addition, a method of detecting the presence of any one of the polypeptides according to the invention in a sample, said method comprising:

[0128] a) bringing the sample to be tested into contact with an antibody directed against 1) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-8, 2) a polypeptide fragment or variant of a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 5-8, or 3) a polypeptide termed “homologous” to a polypeptide comprising amino acid sequence of any one of SEQ ID NOS: 5-8; and

[0129] b) detecting the antigen/antibody complex formed.

[0130] The invention also relates to a box or kit for diagnosis or for detecting the presence of any one of polypeptide in accordance with the invention in a sample, said box comprising:

[0131] a) an antibody directed against 1) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-8, 2) a polypeptide fragment or variant of a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-8, or 3) a polypeptide “homologous” to a polypeptide comprising amino acid sequence of SEQ ID NOS: 5-8; and

[0132] b) a reagent allowing the detection of the antigen/antibody complexes formed.

[0133] The invention also relates to a pharmaceutical composition comprising a nucleic acid according to the invention.

[0134] The invention also provides compositions comprising a nucleic acid encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides according to the invention and compositions comprising any one of the ABCA5, ABCA6, ABCA9, ABCA10 polypeptides according to the invention intended for the treatment of diseases linked to a deficiency of cholesterol reverse transport or inflammatory lipophilic substances transport.

[0135] The present invention also relates to a new therapeutic approach for the treatment of pathologies linked to a deficiency of cholesterol reverse transport or inflammatory lipophilic substances transport, comprising transferring and expressing in vivo nucleic acids encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins according to the invention.

[0136] Thus, the present invention offers a new approach to the treatment and/or prevention of pathologies linked to the abnormalities of cholesterol reverse transport or inflammatory lipophilic substances. Specifically, the present invention provides methods to restore or promote improved cholesterol reverse transport or improved inflammatory lipophilic substances transport in a patient or subject.

[0137] Consequently, the invention also relates to a composition intended for the prevention and/or treatment of subjects affected by a dysfunction of cholesterol reverse transport or inflammatory lipophilic substances transport, comprising a nucleic acid encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins in combination with one or more physiologically-compatible vehicle and/or excipient.

[0138] According to one embodiment of the invention, a composition is provided for the in vivo production of any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins. This composition comprises a nucleic acid encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides placed under the control of appropriate regulatory sequences in solution in a physiologically-compatible vehicle and/or excipient.

[0139] Therefore, the present invention also relates to a composition comprising a nucleic acid encoding a polypeptide comprising an amino acid sequence of any one of SEQ ID NOS: 5-8, wherein the nucleic acid is placed under the control of appropriate regulatory elements.

[0140] Such a composition may comprise a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOS: 1-4 and 9-126, placed under the control of appropriate regulatory elements.

[0141] The invention also relates to a composition intended for the prevention of or treatment of subjects affected by a dysfunction of cholesterol reverse transport or inflammatory lipophilic substances transport, comprising a recombinant vector according to the invention in combination with one or more physiologically-compatible vehicle and/or excipient.

[0142] According to another aspect, the subject of the invention is also a preventive or curative therapeutic method of treating diseases caused by a deficiency of cholesterol reverse transport or inflammatory lipophilic substances transport, such a method comprising administering to a patient a nucleic acid encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides according to the invention, said nucleic acid being combined with one or more physiologically-appropriate vehicles and/or excipients.

[0143] The invention relates to a composition for the prevention and/or treatment of a patient or subject affected by a dysfunction of cholesterol reverse transport or inflammatory lipophilic substances transport, comprising a therapeutically effective quantity of a polypeptide having an amino acid sequence selected from SEQ ID NOS: 5-8 combined with one or more physiologically-appropriate vehicles and/or excipients.

[0144] According to one embodiment, a method of introducing at least a nucleic acid according to the invention into a host cell, for example, a host cell obtained from a mammal, in vivo, comprises a step during which a preparation comprising a pharmaceutically compatible vector and a “naked” nucleic acid according to the invention, placed under the control of appropriate regulatory sequences, is introduced by local injection at the site of the chosen tissue, for example, a smooth muscle tissue, the “naked” nucleic acid being absorbed by the cells of this tissue.

[0145] According to yet another aspect, the subject of the invention is also a preventive or curative therapeutic method of treating diseases caused by a deficiency of cholesterol reverse transport or inflammatory lipophilic substances transport, such a method comprising administering to a patient a therapeutically effective quantity of at least one the ABCA5, ABCA6, ABCA9, or ABCA10 polypeptides according to the invention, said polypeptide being combined with one or more physiologically-appropriate vehicles and/or excipients.

[0146] The invention also provides methods for screening small molecules and compounds that act on any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins to identify agonists and antagonists of such polypeptides that can restore or promote improved cholesterol reverse transport or inflammatory lipophilic substances transport to effectively cure and or prevent dysfunctions thereof. These methods are useful for identifying small molecules and compounds for therapeutic use in the treatment of diseases due to a deficiency of cholesterol reverse transport or inflammatory lipophilic substances transport.

[0147] The invention also relates to the use of any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides or a cell expressing any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides according to the invention for screening active ingredients for the prevention and/or treatment of diseases resulting from a dysfunction cholesterol reverse transport or inflammatory lipophilic substances transport.

[0148] The invention also relates to a method of screening a compound or small molecule, an agonist or antagonist of any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides, said method comprising:

[0149] a) preparing a membrane vesicle comprising any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides and a lipid substrate comprising a detectable marker;

[0150] b) incubating the vesicle obtained in step a) with an agonist or antagonist candidate compound;

[0151] c) qualitatively and/or quantitatively measuring release of the lipid substrate comprising a detectable marker; and

[0152] d) comparing the release measurement obtained in step c) with a measurement of release of a labelled lipid substrate by a vesicle that has not been previously incubated with the agonist or antagonist candidate compound.

[0153] In a one embodiment of this method, the ABCA5,ABCA6,ABCA9, and ABCA10 polypeptides comprise SEQ ID NOS: 5-8, respectively.

[0154] The invention also relates to a method of screening a compound or small molecule, an agonist or antagonist of any one of ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides, said method comprising:

[0155] a) obtaining a cell, for example, a cell line, that, either naturally or after transfecting the cell with any one of the ABCA5, ABCA6, ABCA9, and ABCA10 encoding nucleic acids, expresses the ABCA5, ABCA6, ABCA9, or ABCA10 polypeptide;

[0156] b) incubating the cell of step a) in the presence of an anion labelled with a detectable marker;

[0157] c) washing the cell of step b) in order to remove the excess of the labelled anion which has not penetrated into these cells;

[0158] d) incubating the cell obtained in step c) with an agonist or antagonist candidate compound for the any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides;

[0159] e) measuring efflux of the labelled anion; and

[0160] f) comparing the value of efflux of the labelled anion determined in step e) with the value of efflux of a labelled anion measured with a cell that has not been previously incubated in the presence of the agonist or antagonist candidate compound for any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides.

[0161] In one embodiment of this method, the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides comprise SEQ ID NOS: 5-8, respectively.

[0162] The invention also relates to a method of screening a compound or small molecule, an agonist or antagonist of any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides, said method comprising:

[0163] a) culturing cells of a human monocytic line in an appropriate culture medium, in the presence of purified human albumin;

[0164] b) incubating the cells of step a) simultaneously in the presence of a compound stimulating the production of IL-1 beta and of the agonist or antagonist candidate compound;

[0165] c) incubating the cells obtained in step b) in the presence of an appropriate concentration of ATP;

[0166] d) measuring IL-1 beta released into the cell culture supernatant; and

[0167] e) comparing the value of the release of the IL-1 beta obtained in step d) with the value of the IL-1 beta released into the culture supernatant of cells that have not been previously incubated in the presence of the agonist or antagonist candidate compound.

BRIEF DESCRIPTION OF THE DRAWINGS

[0168]FIG. 1: represents the Map of the 17q24 region containing the ABCA5, 6, 9, and 10 genes. A physical map of the portion of chromosome 17q24 is shown containing the 5 ABCA genes. Location of the microsatellite marker D17S940 locus is indicated along with the boundaries of BAC clones hRPK.235_I_(—)10 and hRPK.293_K_(—)20 (GenBank accession #s AC005495, AC005922). Gene orientation is indicated by the arrows, and the size and location of the corresponding transcripts is shown below the map. 0 indicates the initiation codon; | represents the stop codon;—symbolizes the working draft sequences.

[0169]FIG. 2: represents the alignment of ABC1-like genes. An alignment of the amino acid sequence of the full-length ABCA6, 8, and 9 open reading frames and the partial sequence of ABCA5 is shown as aligned to ABCA1.

[0170]FIG. 3: Maximum parsimony tree of ABC1-like genes. Phylogenetic trees were constructed with the alignment of the N- and C-terminal ATP-binding domains' sequence by both neighbor joining and maximum parsimony methods.

[0171]FIG. 4: Northern blot analysis of poly(A)+ RNA from 20 human tissues: pancreas (lane 1), kidney (2), skeletal muscle (3), liver (4), lung (5), placenta (6), brain (7), heart (8), leukocyte (9), colon (10), small intestine (11), ovary (12), testis (13), prostate (14), thymus (15), spleen (16), fetal kidney (17), fetal liver (18), fetal lung (19) and fetal brain (20). Hybridization was with a probe specific for either ABCA5 (A), ABCA6 (B), ABCA9 (C), or ABCA10 (D).

[0172]FIG. 5: displays the pattern of expression of the gene encoding the ABCA9 protein by in situ hybridization using an antisense ABCA9 RNA probe on a normal renal artery section showing medial smooth muscle (60X).

[0173]FIG. 6: displays the pattern of expression of the gene encoding the ABCA9 protein by in situ hybridization using an antisense ABCA9 RNA probe on a normal renal artery section showing adventitial nerve and Schwann cells (60X).

[0174]FIG. 7: displays the pattern of expression of the gene encoding the ABCA9 protein by in situ hybridization using an antisense ABCA9 RNA probe on a normal renal artery section showing adjacent ganglions and Schwann cells (60X).

[0175]FIG. 8: displays the pattern of expression of the gene encoding the ABCA9 protein by in situ hybridization using an antisense ABCA9 RNA probe on an adjacent kidney section showing a collecting duct epithelium (60X).

[0176]FIG. 9: displays the pattern of expression of the gene encoding the ABCA9 protein by in situ hybridization using an antisense ABCA9 RNA probe on an adjacent kidney section showing a renal tubular epithelial (60X).

[0177]FIG. 10: displays the pattern of expression of the gene encoding the ABCA9 protein by in situ hybridization using an antisense ABCA9 RNA probe on a section of normal heart showing cardiac myocytes (60X).

[0178]FIG. 11: displays the pattern of expression of the gene encoding the ABCA9 protein by in situ hybridization using an antisense ABCA9 RNA probe on a section of normal heart showing interstitial vascular endothelial cells and fibroblasts (60X).

[0179]FIG. 12: displays the pattern of expression of the gene encoding the ABCA10 protein by in situ hybridization using an antisense ABCA10 RNA probe on a section of arterial tissues showing an adjacent lymph node, lymphocytes and macrophages (60X).

[0180]FIG. 13: displays the pattern of expression of the gene encoding the ABCA10 protein by in situ hybridization using an antisense ABCA10 RNA probe on a section of arterial tissues showing Schwann cells in a nerve (60X).

[0181]FIG. 14: displays the pattern of expression of the gene encoding the ABCA10 protein by in situ hybridization using an antisense ABCA10 RNA probe on a myocardial tissue section showing myointimal cells in an atheroma (60X).

[0182]FIG. 15: displays the pattern of expression of the gene encoding the ABCA10 protein by in situ hybridization using an antisense ABCA10 RNA probe on an tissue section adjacent to a myocardial tissue, showing a ganglion and Schwann cells (60X).

[0183]FIG. 16: displays the pattern of expression of the gene encoding the ABCA10 protein by in situ hybridization using an antisense ABCA10 RNA probe on a skeletal tissue section showing macrophages (60X).

[0184]FIG. 17: displays the pattern of expression of the gene encoding the ABCA10 protein by in situ hybridization using an antisense ABCA10 RNA probe on a skeletal tissue section showing Schwann cells in a nerve (60X).

DETAILED DESCRIPTION OF THE INVENTION

[0185] General Definitions

[0186] The present invention encompasses the isolation of human genes encoding the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides of the invention, including full length or naturally occurring forms of ABCA5, ABCA6, ABCA9, and ABCA10 and any antigenic fragments thereof from any animal, including mammals, for example humans, and birds.

[0187] In accordance with the present invention, conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art are used. Such techniques are fully explained in the literature (Sambrook et al., 1989. Molecular cloning a laboratory manual. 2ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Glover, 1985, DNA Cloning: A practical approach, volumes I and II oligonucleotide synthesis, MRL Press, LTD., Oxford, U.K.; Hames and Higgins, 1985, Transcription and translation; Hames and Higgins, 1984, Animal Cell Culture; Freshney, 1986, Immobilized Cells And Enzymes, IRL Press; and Perbal, 1984, A practical guide to molecular cloning).

[0188] As used herein, the term “gene” refers to an assembly of nucleotides that encode a polypeptide and includes cDNA and genomic DNA nucleic acids.

[0189] The term “isolated” for the purposes of the present invention refers to a biological material (nucleic acid or protein) that has been removed from its original environment (the environment in which it is naturally present). For example, a polynucleotide present in the natural state in a plant or in an animal is not isolated. The same polynucleotide separated from the adjacent nucleic acids in which it is naturally inserted in the genome of the plant or animal is considered as being “isolated”.

[0190] An “isolated” polynucleotide may be included in a vector and/or such a polynucleotide may be included in a composition and remain nevertheless in the isolated state because of the fact that the vector or the composition does not constitute its natural environment.

[0191] The term “purified” does not require the material to be present in a form exhibiting absolute purity exclusive of the presence of other compounds. It is a relative definition. A polynucleotide is in the “purified” state after purification from the starting material or from the natural material by at least one order of magnitude.

[0192] For the purposes of the present description, the expression “nucleotide sequence” is used to designate either a polynucleotide or a nucleic acid. The expression “nucleotide sequence” covers the genetic material itself and is therefore not restricted to the information relating to its sequence.

[0193] The terms “nucleic acid”, “polynucleotide”, “oligonucleotide” or “nucleotide sequence” encompass RNA, DNA, or cDNA sequences, and RNA/DNA hybrid sequences of more than one nucleotide, either in the single-stranded form or in the duplex, double-stranded form.

[0194] A “nucleic acid” is a polymeric compound comprised of covalently-linked subunits called nucleotides. The term “nucleic acid” includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single-stranded or double-stranded. DNA includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA. The sequence of nucleotides that encodes a protein is called the sense sequence or coding sequence.

[0195] The term “nucleotide” designates both the natural nucleotides (A, T, G, C) as well as modified nucleotides that comprise at least one modification such as (1) an analog of a purine, (2) an analog of a pyrimidine, or (3) an analogous sugar, examples of such modified nucleotides are described, for example, in the PCT application No. WO 95/04 064.

[0196] For the purposes of the present invention, a first polynucleotide is considered as being “complementary” to a second polynucleotide when each base of the first nucleotide is paired with the complementary base of the second polynucleotide whose orientation is reversed. The complementary bases are A and T (or A and U), or C and G.

[0197] “Heterologous” DNA refers to DNA not naturally located in the cell or in a chromosomal site of the cell. The heterologous DNA may include a gene foreign to the cell.

[0198] As used herein, the term “homologous” in all its grammatical forms and spelling variations refers to the relationship between proteins that possess a “common evolutionary origin,” including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc.) (Reeck et al., 1987, Cell 50 :667)). Such proteins (and their encoding genes) have sequence homology, as reflected by their high degree of sequence similarity.

[0199] Accordingly, the term “sequence similarity” in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al., supra). However, in common usage and in the instant application, the term “homologous,” when modified with an adverb such as “highly,” may refer to sequence similarity and not a common evolutionary origin.

[0200] For example, two DNA sequences are “substantially homologous” or “substantially similar” when at least about 50% (preferably at least about 75%, and more preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; Glover et al. (1985. DNA Cloning: A practical approach, volumes I and II oligonucleotide synthesis, MRL Press, Ltd, Oxford, U.K.); Hames and Higgins (1985. Transcription and Translation).

[0201] Similarly, two amino acid sequences are “substantially homologous” or “substantially similar” when greater than 30% of the amino acids are identical, or greater than about 60% are similar (functionally identical). Preferably, the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) pileup program.

[0202] The “percentage identity” between two nucleotide or amino acid sequences, for the purposes of the present invention, may be determined by comparing two sequences aligned optimally through a window for comparison.

[0203] The portion of the nucleotide or polypeptide sequence in the window for comparison may thus comprise additions or deletions (for example “gaps”) relative to the reference sequence (which does not comprise these additions or these deletions) so as to obtain an optimum alignment of the two sequences.

[0204] The percentage identity is calculated by determining the number of positions at which an identical nucleic base or an identical amino acid residue is observed for the two sequences (nucleic or peptide) compared, dividing the number of positions at which there is identity between the two bases or amino acid residues by the total number of positions in the window for comparison, and then multiplying the result by 100 in order to obtain the percentage sequence identity.

[0205] The optimum sequence alignment for the comparison may be achieved using a computer with the aid of known algorithms contained in the package from the company Wisconsin Genetics Software Package, Genetics Computer Group (Gcg), 575 Science Doctor, Madison, Wis.

[0206] By way of illustration, it will be possible to produce the percentage sequence identity with the aid of the BLAST software (versions BLAST 1.4.9 of March 1996, BLAST 2.0.4 of February 1998 and BLAST 2.0.6 of September 1998), using exclusively the default parameters (Altschul et al, 1990, Mol. Biol., 215:403-410; Altschul et al, 1997, Nucleic Acids Res., 25:3389-3402). Blast searches for sequences similar/homologous to a reference “request” sequence, with the aid of the Altschul et al. algorithm. The request sequence and the databases used may be of the peptide or nucleic types, any combination being possible.

[0207] The term “corresponding to” is used herein to refer to similar or homologous sequences, whether the exact position is identical or different from the molecule to which the similarity or homology is measured. A nucleic acid or amino acid sequence alignment may include spaces. Thus, the term “corresponding to” refers to the sequence similarity and not to the numbering of the amino acid residues or nucleotide bases.

[0208] A gene encoding any one of the ABCA5, ABCA6, ABCA9 and ABCA10 polypeptides of the invention, whether genomic DNA or cDNA, can be isolated from any source, for example, from a human cDNA or genomic library. Methods for obtaining genes are well known in the art as described above (see, e.g., Sambrook et al., 1989, Molecular cloning: a laboratory manual 2ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

[0209] Accordingly, any animal cell can serve as the nucleic acid source for the molecular cloning of any one of the ABCA5, ABCA6, ABCA9, and ABCA10 genes. The DNA may be obtained by standard procedures known in the art from cloned DNA (e.g., a DNA “library”) and preferably is obtained from a cDNA library prepared from tissues with high level expression of the protein and/or the transcripts, by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell (See, for example, Sambrook et al., 1989, Molecular cloning: a laboratory manual. 2ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Glover, 1985, DNA Cloning: A Practical Approach, Volumes I and II Oligonucleotide Synthesis, MRL Press, Ltd., Oxford, U.K).

[0210] Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will not contain intron sequences. Whatever the source, the gene should be molecularly cloned into a suitable vector for propagation of the gene.

[0211] In the molecular cloning of the gene from genomic DNA, DNA fragments are generated, some of which will encode the desired gene. The DNA may be cleaved at specific sites using various restriction enzymes. Alternatively, one may use DNAse in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication. The linear DNA fragments can then be separated according to size by standard techniques, including, but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography.

[0212] Once the DNA fragments are generated, identification of the specific DNA fragment containing the desired ABCA5, ABCA, ABCA9, and ABCA10 genes may be accomplished in a number of ways. For example, if an amount of a portion of one of ABCA5, ABCA6, ABCA9, and ABCA10 genes or its specific RNA, or a fragment thereof, is available and can be purified and labelled, the generated DNA fragments may be screened by nucleic acid hybridization to the labelled probe (Benton and Davis, Science (1977), 196:180; Grunstein et al., Proc.Natl. Acad. Sci. U.S.A. (1975) 72:3961). For example, a set of oligonucleotides corresponding to the partial coding sequence information obtained for the ABCA5, ABCA6, ABCA9, ABCA10 proteins can be prepared and used as probes for DNA encoding any one of the ABCA5, ABCA6, ABCA9 and ABCA10 genes, as was done in a specific example, infra, or as primers for cDNA or mRNA synthesis (e.g., in combination with a poly-T primer for RT-PCR). Preferably, a fragment is selected that is highly unique to one of the ABCA5, ABCA6, ABCA9, and ABCA10 nucleic acids or polypeptides of the invention. Those DNA fragments with substantial homology to the probe will hybridize. As noted above, the greater the degree of homology, the more stringent hybridization conditions can be used. In one embodiment, various stringency hybridization conditions are used to identify homologous ABCA5, ABCA6, ABCA9, and ABCA10 genes.

[0213] Further selection can be carried out on the basis of the properties of the gene, e.g., if the gene encodes a protein product having the isoelectric, electrophoretic, amino acid composition, or partial amino acid sequence of one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins as disclosed herein. Thus, the presence of the gene may be detected by assays based on the physical, chemical, or immunological properties of its expressed product. For example, cDNA clones or DNA clones which hybrid-select the proper mRNAs can be selected which produce a protein that, e.g., has similar or identical electrophoretic migration, isoelectric focusing, or non-equilibrium pH gel electrophoresis behaviour, proteolytic digestion maps, or antigenic properties as known for ABCA5, ABCA6, ABCA9, and ABCA10.

[0214] The ABCA5, ABCA6, ABCA9, and ABCA10 genes of the invention may also be identified by mRNA selection, i.e., by nucleic acid hybridization followed by in vitro translation. According to this procedure, nucleotide fragments are used to isolate complementary mRNAs by hybridization. Such DNA fragments may represent available, purified ABCA5, ABCA6, ABCA9, ABCA10 DNAs or may be synthetic oligonucleotides designed from the partial coding sequence information. Immunoprecipitation analysis or functional assays (e.g., tyrosine phosphatase activity) of the in vitro translation products of the products of the isolated mRNAs identifies the mRNA and, therefore, the complementary DNA fragments that contain the desired sequences. In addition, specific mRNAs may be selected by adsorption of polysomes isolated from cells to immobilized antibodies specifically directed against any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides of the invention.

[0215] Radiolabeled ABCA5, ABCA6, ABCA 9, and ABCA10 cDNAs can be synthesized using the selected mRNA (from the adsorbed polysomes) as a template. The radiolabeled mRNA or cDNA may then be used as a probe to identify homologous ABCA5, ABCA6, ABCA9, and ABCA10 DNA fragments from among other genomic DNA fragments.

[0216] “Variant” of a nucleic acid according to the invention will be understood to mean a nucleic acid that differs by one or more bases relative to the reference polynucleotide. A variant nucleic acid may be of natural origin, such as an allelic variant which exists naturally, or it may be a nonnatural variant obtained, for example, by mutagenic techniques.

[0217] In general, the differences between the reference (generally, wild-type) nucleic acid and the variant nucleic acid are small such that the nucleotide sequences of the reference nucleic acid and of the variant nucleic acid are very similar and, in many regions, identical. The nucleotide modifications present in a variant nucleic acid may be silent, which means that they do not alter the amino acid sequences encoded by said variant nucleic acid. However, the changes in nucleotides in a variant nucleic acid may also result in substitutions, additions, or deletions in the polypeptide encoded by the variant nucleic acid in relation to the polypeptides encoded by the reference nucleic acid. In addition, nucleotide modifications in the coding regions may produce conservative or non-conservative substitutions in the amino acid sequence of the polypeptide.

[0218] Preferably, the variant nucleic acids according to the invention encode polypeptides that substantially conserve the same function or biological activity as the polypeptide of the reference nucleic acid or, alternatively, the capacity to be recognized by antibodies directed against the polypeptides encoded by the initial reference nucleic acid. Some variant nucleic acids will thus encode mutated forms of the polypeptides whose systematic study will make it possible to deduce structure-activity relationships of the proteins in question. Knowledge of these variants in relation to the disease studied is essential since it makes it possible to understand the molecular cause of the pathology.

[0219] “Fragment” will be understood to mean a nucleotide sequence of reduced length relative to the reference nucleic acid and comprising, over the common portion, a nucleotide sequence identical to the reference nucleic acid. Such a nucleic acid “fragment” according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent. Such fragments comprise or, alternatively, consist of, oligonucleotides ranging in length from 8, 10, 12, 15, 18, 20 to 25, 30, 40, 50, 70, 80, 100, 200, 500, 1000 or 1500 consecutive nucleotides of a nucleic acid according to the invention.

[0220] A “nucleic acid molecule” refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A “recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.

[0221] A nucleic acid molecule is “hybridizable” to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al., supra). The conditions of temperature and ionic strength determine the “stringency” of the hybridization. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a T_(m) of 55°, can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5% SDS. Moderate stringency hybridization conditions correspond to a higher T_(m), e.g., 40% formamide, with 5× or 6×SCC. High stringency hybridization conditions correspond to the highest T_(m), e.g., 50% formamide, 5× or 6×SCC. Hybridization requires that the two nucleic acids contain complementary sequences, although, depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T_(m) for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher T_(m)) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating T_(m) have been derived (see Sambrook et al., supra). For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra). The minimum length for a hybridizable nucleic acid may be at least about 10 nucleotides; at least about 15 nucleotides; or at least about 20 nucleotides.

[0222] In one embodiment, the term “standard hybridization conditions” refers to a T_(m) of 55° C., and utilizes conditions as set forth above. In another embodiment, the T_(m) is 60° C.; in another embodiment, the T_(m) is 65° C.

[0223] “High stringency hybridization conditions” for the purposes of the present invention will be understood to mean the following conditions:

[0224] 1-Membrane competition and PREHYBRIDIZATION:

[0225] Mix: 40 μl salmon sperm DNA (10 mg/ml)

[0226] +40 μl human placental DNA (10 mg/ml)

[0227] Denature for 5 minutes at 96° C., then immerse the mixture in ice.

[0228] Remove the 2×SSC and pour 4 ml of formamide mix in the hybridization tube containing the membranes.

[0229] Add the mixture of the two denatured DNAs.

[0230] Incubation at 42° C. for 5 to 6 hours, with rotation.

[0231] 2-Labeled probe competition:

[0232] Add to the labeled and purified probe 10 to 50 μl Cot I DNA, depending on the quantity of repeats.

[0233] Denature for 7 to 10 minutes at 95° C.

[0234] Incubate at 65° C. for 2 to 5 hours.

[0235] 3-HYBRIDIZATION:

[0236] Remove the prehybridization mix.

[0237] Mix 40 μl salmon sperm DNA +40 μl human placental DNA; denature for 5 min at 96° C., then immerse in ice.

[0238] Add to the hybridization tube 4 ml of formamide mix, the mixture of the two DNAs and the denatured labeled probe/Cot I DNA.

[0239] Incubate 15 to 20 hours at 42° C., with rotation.

[0240] 4-Washes and Exposure:

[0241] One wash at room temperature in 2×SSC, to rinse.

[0242] Wash twice 5 minutes at room temperature 2×SSC and 0.1% SDS at 65° C.

[0243] Wash twice 15 minutes 0.1×SSC and 0.1% SDS at 65° C.

[0244] Enclose the membranes in clear plastic wrap and expose.

[0245] The hybridization conditions described above are adapted to hybridization, under high stringency conditions, of a molecule of nucleic acid of varying length from 20 nucleotides to several hundreds of nucleotides. It goes without saying that the hybridization conditions described above may be adjusted as a function of the length of the nucleic acid whose hybridization is sought or of the type of labeling chosen, according to techniques known to one skilled in the art. Suitable hybridization conditions may, for example, be adjusted according to the teaching contained in the manual by Hames and Higgins (1985, supra).

[0246] As used herein, the term “oligonucleotide” refers to a nucleic acid, generally of at least 15 nucleotides, that is hybridizable to a nucleic acid according to the invention. Oligonucleotides can be labelled, e.g., with ³²P-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated. In one embodiment, a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid encoding an ABCA5-6, 9-10 polypeptide of the invention. In another embodiment, oligonucleotides (one or both of which may be labelled) can be used as PCR primers, either for cloning full lengths or fragments of any one of the ABCA5, ABCA6, ABCA9,and ABCA10 nucleic acids or to detect the presence of nucleic acids encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 genes. In a further embodiment, an oligonucleotide of the invention can form a triple helix with any one of the ABCA5, ABCA6, ABCA9, and ABCA10 DNA molecules. Generally, oligonucleotides are prepared synthetically, for example, on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.

[0247] “Homologous recombination” refers to the insertion of a foreign DNA sequence of a vector in a chromosome. Preferably, the vector targets a specific chromosomal site for homologous recombination. For specific-specific homologous recombination, the vector will contain sufficiently long regions of homology to sequences of the chromosome to allow complementary binding and incorporation of the vector into the chromosome. Longer regions of homology and greater degrees of sequence similarity may increase the efficiency of homologous recombination.

[0248] A DNA “coding sequence” is a double-stranded DNA sequence that is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence.

[0249] Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell. In eukaryotic cells, polyadenylation signals are control sequences.

[0250] “Regulatory region” means a nucleic acid sequence which regulates the expression of a nucleic acid. A regulatory region may include sequences which are naturally responsible for expressing a particular nucleic acid (a homologous region) or may include sequences of a different origin (responsible for expressing different proteins or even synthetic proteins). In particular, the sequences can be sequences of eukaryotic or viral genes or derived sequences that stimulate or repress transcription of a gene in a specific or non-specific manner and in an inducible or non-inducible manner. Regulatory regions include origins of replication, RNA splice sites, enhancers, transcriptional termination sequences, signal sequences that direct the polypeptide into the secretory pathways of the target cell, and promoters.

[0251] A regulatory region from a “heterologous source” is a regulatory region that is not naturally associated with the expressed nucleic acid. Included among the heterologous regulatory regions are regulatory regions from a different species, regulatory regions from a different gene, hybrid regulatory sequences, and regulatory sequences that do not occur in nature, but which are designed by one having ordinary skill in the art.

[0252] A “cassette” refers to a segment of DNA that can be inserted into a vector at specific restriction sites. The segment of DNA encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation.

[0253] A “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined, for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

[0254] A coding sequence is “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced and translated into the protein encoded by the coding sequence.

[0255] A “signal sequence” is included at the beginning of the coding sequence of a protein to be expressed on the surface of a cell. This sequence encodes a signal peptide, N-terminal to the mature polypeptide, that directs the host cell to translocate the polypeptide. The term “translocation signal sequence” is used herein to refer to this sort of signal sequence. Translocation signal sequences can be found associated with a variety of proteins native to eukaryotes and prokaryotes, and are often functional in both types of organisms.

[0256] A “polypeptide” is a polymeric compound comprised of covalently linked amino acid residues. Amino acids have the following general structure:

[0257] Amino acids are classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxyl (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, in which the side chain is fused to the amino group.

[0258] A “protein” is a polypeptide that plays a structural or functional role in a living cell.

[0259] The polypeptides and proteins of the invention may be glycosylated or unglycosylated.

[0260] “Homology” means similarity of sequence reflecting a common evolutionary origin. Polypeptides or proteins are said to have homology, or similarity, if a substantial number of their amino acids are either (1) identical, or (2) have a chemically similar R side chain. Nucleic acids are said to have homology if a substantial number of their nucleotides are identical.

[0261] “Isolated polypeptide” or “isolated protein” is a polypeptide or protein that is substantially free of those compounds that are normally associated therewith in its natural state (e.g., other proteins or polypeptides, nucleic acids, carbohydrates, lipids). “Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with biological activity, and which may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into a pharmaceutically acceptable preparation.

[0262] “Fragment” of a polypeptide according to the invention will be understood to mean a polypeptide whose amino acid sequence is shorter than that of the reference polypeptide and that comprises, over the entire portion with these reference polypeptides, an identical amino acid sequence. Such fragments may, where appropriate, be included in a larger polypeptide of which they are a part. Such fragments of a polypeptide according to the invention may have a length of about 5, about 10, about 15, about 20, about 30 to about 40, about 50, about 100, about 200 or about 300 amino acids.

[0263] “Variant” of a polypeptide according to the invention will be understood to mean mainly a polypeptide whose amino acid sequence contains one or more substitutions, additions, or deletions of at least one amino acid residue, relative to the amino acid sequence of the reference polypeptide, it being understood that the amino acid substitutions may be either conservative or nonconservative.

[0264] A “variant” of a polypeptide or protein is any analogue, fragment, derivative, or mutant that is derived from a polypeptide or protein and that retains at least one biological property of the polypeptide or protein. Different variants of the polypeptide or protein may exist in nature. These variants may result from allelic variations characterized by differences in the nucleotide sequences of the structural gene coding for the protein or may involve differential splicing or post-translational modification. Variants also include related proteins having substantially the same biological activity, but obtained from a different species.

[0265] The skilled artisan can produce variants having single or multiple amino acid substitutions, deletions, additions, or replacements. These variants may include, inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to the polypeptide or protein, (c) variants in which one or more of the amino acids includes a substituent group, and (d) variants in which the polypeptide or protein is fused with another polypeptide such as serum albumin. The techniques for obtaining these variants, including genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques, are known to persons having ordinary skill in the art.

[0266] If such allelic variations, analogues, fragments, derivatives, mutants, and modifications, including alternative mRNA splicing forms and alternative post-translational modification forms, result in derivatives of the polypeptide that retain any of the biological properties of the polypeptide, they are intended to be included within the scope of this invention.

[0267] A “vector” is a replicon, such as plasmid, virus, phage, or cosmid to which another DNA segment may be attached so as to bring about the replication of the attached segment. A “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., is capable of replication under its own control.

[0268] The present invention also relates to cloning vectors containing genes encoding analogs and derivatives any of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides of the invention that have the same or homologous functional activity as that of ABCA5, ABCA6, ABCA9, ABCA10 polypeptides and tp homologs thereof from other species. The production and use of derivatives and analogs related to the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides are within the scope of the present invention. In general, The derivatives or analogs are functionally active, i.e., capable of exhibiting one or more functional activities associated with the full-length, wild-type ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides of the invention.

[0269] ABCA5, ABCA6, ABCA9, and ABCA10 derivatives can be made by altering encoding nucleic acid sequences by substitutions, additions or deletions that provide for functionally equivalent molecules. Preferably, derivatives are made that have enhanced or increased functional activity relative to native ABCA5, ABCA6, ABCA9, and ABCA10. Alternatively, such derivatives may encode soluble fragments of the ABCA5, ABCA6, ABCA9, and ABCA10 extracellular domains that have the same or greater affinity for the natural ligand of ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides of the invention. Such soluble derivatives may be potent inhibitors of ligand binding to ABCA5, ABCA6, ABCA9, and ABCA10.

[0270] Due to the degeneracy of nucleotide coding sequences, other DNA sequences that encode substantially the same amino acid sequences as that of the ABCA5, ABCA6, ABCA9, and ABCA10 genes may be used in the practice of the present invention. These include, but are not limited to, allelic genes, homologous genes from other species, and nucleotide sequences comprising all or portions of ABCA5, ABCA6, ABCA9, and ABCA10 genes that are altered by the substitution of different codons that encode the same amino acid residue within the sequence, thereby producing a silent change. Likewise, the ABCA5, ABCA6, ABCA9, and ABCA10 derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins, including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a conservative amino acid substitution. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Amino acids containing aromatic ring structures are phenylalanine, tryptophan, and tyrosine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Subtituition of one amino acid within a group for another is not expected to affect apparent molecular weight as determined by polyacrylamide gel electrophoresis, or isoelectric point.

[0271] Such substitutions include:

[0272] Lys for Arg and vice versa, such that a positive charge may be maintained;

[0273] Glu for Asp and vice versa, such that a negative charge may be maintained;

[0274] Ser for Thr, such that a free —OH can be maintained; and

[0275] Gln for Asn, such that a free CONH₂ can be maintained.

[0276] Amino acid substitutions may also be introduced to substitute an amino acid with a particularly desirable property. For example, a Cys may be introduced as a potential site for disulfide bridge formation with another Cys. A His may be introduced as a particularly “catalytic” site (i.e., His can act as an acid or base and is the most common amino acid in biochemical catalysis). Pro may be introduced because of its particularly planar structure, which induces b-turns in the protein's structure.

[0277] The genes encoding ABCA5, ABCA6, ABCA9, and ABCA10 derivatives and analogs of the invention can be produced by various methods known in the art. The manipulations that result in their production can occur at the gene or protein level. For example, the cloned ABCA5, ABCA6, ABCA9, and ABCA10 sequences can be modified by any of numerous strategies known in the art (Sambrook et al., 1989, supra). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro. Production of a gene encoding a derivative or analog of any one of the ABCA5, ABCA6, ABCA9, and ABCA10 should ensure that the modified gene remains within the same translational reading frame as the ABCA5, ABCA6, ABCA9, and ABCA10 genes, uninterrupted by translational stop signals in the region where the desired activity is encoded.

[0278] Additionally, the ABCA5, ABCA6, ABCA9, and ABCA10-encoding nucleic acids can be mutated in vitro or in vivo to create and/or destroy translation, initiation, and/or termination sequences or to create variations in coding regions and/or form new restriction endonuclease sites or destroy pre-existing ones to facilitate further in vitro modification. Such mutations may enhance the functional activity of the mutated ABCA5, ABCA6, ABCA9, and ABCA10 genes products. Any technique for mutagenesis known in the art may be used, including, inter alia, in vitro site-directed mutagenesis (Hutchinson et al., (1978) Biol. Chem. 253:6551; Zoller and Smith, (1984) DNA, 3:479-488; Oliphant et al., (1986) Gene 44:177; Hutchinson et al., (1986) Proc. Natl. Acad. Sci. U.S.A. 83:710; Huygen et al., (1996) Nature Medicine, 2(8):893-898) and use of TAB® linkers (Pharmacia). PCR techniques are preferred for site-directed mutagenesis (Higuchi, 1989, “Using PCR to Engineer DNA”, in PCR Technology: Principles and Applications for DNA Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70).

[0279] Identified and isolated ABCA5, ABCA6, ABCA9, and ABCA10 genes may then be inserted into an appropriate cloning vector. A large number of vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Examples of vectors for Escherichia coli include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as pBR322 derivatives or pUC plasmid derivatives, e.g., PGEX vectors, pmal-c, pFLAG, etc. The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector that has complementary cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically modified. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated. The cloned gene may be contained on a shuttle vector plasmid, which provides for expansion in a cloning cell, e.g., Escherichia coli, and facile purification for subsequent insertion into an appropriate expression cell line, if such is desired. For example, a shuttle vector, which is a vector that can replicate in more than one type of organism, can be prepared for replication in both Escherichia coli and Saccharomyces cerevisiae by linking sequences from an Escherichia coil plasmid with sequences form the yeast 2m plasmid.

[0280] In an alternative method, the desired gene may be identified and isolated after insertion into a suitable cloning vector in a “shot gun” approach. Enrichment for the desired gene, for example, by size fractionation, can be done before insertion into the cloning vector.

[0281] The nucleotide sequences coding for the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides or antigenic fragments, derivatives, or analogs thereof, or functionally active derivatives, including chimeric proteins thereof, may be inserted into an appropriate expression vector, i.e., a vector that contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. Such elements are termed herein a “promoter.” Thus, nucleic acids encoding the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides of the invention are operationally associated with a promoter in an expression vector of the invention. Both cDNA and genomic sequences can be cloned and expressed under control of such regulatory sequences. An expression vector also usually includes a replication origin.

[0282] The necessary transcriptional and translational signals can be provided on a recombinant expression vector, or they may be supplied by a native gene encoding ABCA5, ABCA6, ABCA9, and ABCA10 and/or its flanking regions.

[0283] Potential host-vector systems include, but are not limited to, mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.

[0284] A recombinant ABCA5, ABCA6, ABCA9, and ABCA10 protein of the invention, or functional fragments, derivatives, chimeric constructs, or analogs thereof, may be expressed chromosomally after integration of the coding sequence by recombination. In this regard, any of a number of amplification systems may be used to achieve high levels of stable gene expression (See Sambrook et al., 1989, supra).

[0285] The cell into which the recombinant vector comprising the nucleic acid encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides according to the invention is cultured in an appropriate cell culture medium under conditions that provide for expression of any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides by the cell.

[0286] Any of the methods previously described for the insertion of DNA fragments into a cloning vector may be used to construct expression vectors containing a gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination (genetic recombination).

[0287] Expression of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression. Promoters that may be used to control ABCA5, ABCA6, ABCA9, and ABCA10 genes expression include, but are not limited to, the SV40 early promoter region (Benoist and Chambon, 1981 Nature 290:304-310), the promoter contained in the 3′ long terminal repeat (LTR) of Rous sarcoma virus (Yamamoto, et al., 1980 Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981 Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982 Nature 296:39-42); prokaryotic expression vectors such as the β-lactamase promoter (Villa-Kamaroff, et al., 1978 Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer, et al., 1983 Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also “Useful proteins from recombinant bacteria” in Scientific American, 1980, 242:74-94; promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter; and the animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region, which is active in pancreatic acinar cells (Swift et al., 1984 Cell 38:639-646; Ornitz et al., 1986 Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987); insulin gene control region, which is active in pancreatic beta cells (Hanahan, 1985 Nature: 315:115-122), immunoglobulin gene control region, which is active in lymphoid cells (Grosschedl et al., 1984 Cell 38:647-658; Adames et al., 1985 Nature 318:533-538; Alexander et al., 1987 Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus control region, which is active in testicular, breast, lymphoid, and mast cells (Leder et al., 1986 Cell 45:485-495), albumin gene control region, which is active in liver (Pinkert et al., 1987 Genes and Devel. 1:268-276), alpha-fetoprotein gene control region, which is active in liver (Krumlauf et al., 1985 Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987 Science 235:53-58), alpha 1-antitrypsin gene control region, which is active in the liver (Kelsey et al., 1987 Genes and Devel. 1:161-171) beta-globin gene control region, which is active in myeloid cells (Mogram et al., 1985 Nature 315:338-340; Kollias et al., 1986 Cell 46:89-94), myelin basic protein gene control region, which is active in oligodendrocyte cells in the brain (Readhead et al., 1987 Cell 48:703-712), myosin light chain-2 gene control region, which is active in skeletal muscle (Sani, 1985 Nature 314:283-286), and gonadotropic releasing hormone gene control region, which is active in the hypothalamus (Mason et al., 1986 Science 234:1372-1378).

[0288] Expression vectors containing a nucleic acid encoding one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides of the invention can be identified by five general approaches: (a) polymerase chain reaction (PCR) amplification of the desired plasmid DNA or specific mRNA, (b) nucleic acid hybridization, (c) presence or absence of selection marker gene functions, (d) analyses with appropriate restriction endonucleases, and (e) expression of inserted sequences. In the first approach, the nucleic acids can be amplified by PCR to provide for detection of the amplified product. In the second approach, the presence of a foreign gene inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted marker gene. In the third approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain “selection marker” gene functions (e.g., β-galactosidase activity, thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of foreign genes in the vector. In another example, if the nucleic acid encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides is inserted within the “selection marker” gene sequence of the vector, recombinants containing ABCA5, ABCA6, ABCA9, and ABCA10 nucleic acids inserts can be identified by the absence of the ABCA5, ABCA6, ABCA9, and ABCA10 gene functions. In the fourth approach, recombinant expression vectors are identified by digestion with appropriate restriction enzymes. In the fifth approach, recombinant expression vectors can be identified by assaying for the activity, biochemical, or immunological characteristics of the gene product expressed by the recombinant, provided that the expressed protein assumes a functionally active conformation.

[0289] A wide variety of host/expression vector combinations may be employed in expressing the nucleic acids of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., Escherichia coli plasmids col EI, pCR1, pBR322, pMaI-C2, pET, pGEX (Smith et al, 1988, Gene 67:31-40), pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e.g., the numerous derivatives of phage 1, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2m plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.

[0290] For example, in a baculovirus expression system, both non-fusion transfer vectors, such as, but not limited to, pVL941 (BamH1 cloning site; Summers), pVL1393 (BamH1, SmaI, XbaI, EcoR1, NotI, XmaIII, BglII, and PstI cloning site; Invitrogen), pVL1392 (BgIII, PstI, NotI, XmaIII, EcoRI, XbaI, SmaI, and BamH1 cloning site; Summers and Invitrogen), and pBlueBacIII (BamH1, BglII, PstI, NcoI, and HindIII cloning site, with blue/white recombinant screening possible; Invitrogen), and fusion transfer vectors, such as, but not limited to, pAc700 (BamH1 and KpnI cloning site, in which the BamH1 recognition site begins with the initiation codon; Summers), pAc701 and pAc702 (same as pAc700, with different reading frames), pAc360 (BamH1 cloning site 36 base pairs downstream of a polyhedrin initiation codon; Invitrogen(195)), and pBlueBacHisA, B, C (three different reading frames, with BamH1, BglII, PstI, NcoI, and HindIII cloning site, an N-terminal peptide for ProBond purification, and blue/white recombinant screening of plaques; Invitrogen (220) can be used.

[0291] Mammalian expression vectors contemplated for use in the invention include vectors with inducible promoters, such as the dihydrofolate reductase (DHFR) promoter, e.g., any expression vector with a DHFR expression vector, or a DHFR/methotrexate co-amplification vector, such as pED (PstI, SalI, SbaI, SmaI, and EcoRI cloning site, with the vector expressing both the cloned gene and DHFR; See, Kaufman, Current Protocols in Molecular Biology, 16.12 (1991). Alternatively, a glutamine synthetase/methionine sulfoximine co-amplification vector, such as pEE14 (HindIII, XbaI, SmaI, SbaI, EcoRI, and BclI cloning site, in which the vector expresses glutamine synthase and the cloned gene; Celltech). In another embodiment, a vector that directs episomal expression under control of Epstein Barr Virus (EBV) can be used, such as pREP4 (BamH1, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII, and KpnI cloning site, constitutive RSV-LTR promoter, hygromycin selectable marker; Invitrogen), pCEP4 (BamH1, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII, and KpnI cloning site, constitutive hCMV immediate early gene, hygromycin selectable marker; Invitrogen), pMEP4 (KpnI, PvuI, NheI, HindIII, NotI, XhoI, SfiI, BamH1 cloning site, inducible methallothionein IIa gene promoter, hygromycin selectable marker: Invitrogen), pREP8 (BamH1, XhoI, NotI, HindIII, NheI, and KpnI cloning site, RSV-LTR promoter, histidinol selectable marker; Invitrogen), pREP9 (KpnI, NheI, HindIII, NotI, XhoI, SfiI, and BamH1 cloning site, RSV-LTR promoter, G418 selectable marker; Invitrogen), and pEBVHis (RSV-LTR promoter, hygromycin selectable marker, N-terminal peptide purifiable via ProBond resin and cleaved by enterokinase; Invitrogen). Selectable mammalian expression vectors for use in the invention include pRc/CMV (HindIII, BstXI, NotI, SbaI, and ApaI cloning site, G418 selection; Invitrogen), pRc/RSV (HindIII, SpeI, BstXI, NotI, XbaI cloning site, G418 selection; Invitrogen), and others. Vaccinia virus mammalian expression vectors (see, Kaufman, 1991, supra) for use according to the invention include, but are not limited to, pSC11 (Smal cloning site, TK- and b-gal selection), pMJ601 (SalI, SmaI, AflI, NarI, BspMII, BamHI, ApaI, NheI, SaclI, KpnI, and HindIII cloning site; TK- and b-gal selection), and pTKgptF1S (EcoRI, PstI, SalI, AccI, HindIII, SbaI, BamH1, and Hpa cloning site, TK or XPRT selection).

[0292] Yeast expression systems can also be used according to the invention to express any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides. For example, the non-fusion pYES2 vector (XbaI, SphI, ShoI, NotI, GstXI, EcoRI, BstXI, BamH1, SacI, Kpn1, and HindIII cloning sit; Invitrogen) or the fusion pYESHisA, B, C (XbaI, SphI, ShoI, NotI, BstXI, EcoRI, BamH1, SacI, KpnI, and HindIII cloning site, N-terminal peptide purified with ProBond resin and cleaved with enterokinase; Invitrogen), to mention just two, can be employed according to the invention.

[0293] Once a particular recombinant DNA molecule is identified and isolated, several methods known in the art may be used to propagate it. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As previously explained, the expression vectors that can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNA vectors, to name but a few.

[0294] In addition, a host cell strain may be chosen that modulates the expression of the inserted sequences or modifies and processes the gene product in the specific fashion desired. Different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, cleavage for example of the signal sequence) of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system can be used to produce a nonglycosylated core protein product. However, the transmembrane ABCA5, ABCA6, ABCA9, and ABCA10 proteins expressed in bacteria may not be properly folded. Expression in yeast can produce a glycosylated product. Expression in eukaryotic cells can increase the likelihood of “native” glycosylation and folding of a heterologous protein. Moreover, expression in mammalian cells can provide a tool for reconstituting, or constituting, ABCA5, ABCA6, ABCA9, and ABCA10 activities. Furthermore, different vector/host expression systems may affect processing reactions, such as proteolytic cleavages, to a different extent.

[0295] Vectors are introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut et al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

[0296] A cell has been “transfected” by exogenous or heterologous DNA when such DNA has been introduced inside the cell. A cell has been “transformed” by exogenous or heterologous DNA when the transfected DNA effects a phenotypic change. In one embodiment of the invention, the transforming DNA is integrated (covalently linked) into the chromosomal DNA making up the genome of the cell.

[0297] A recombinant marker protein expressed as an integral membrane protein can be isolated and purified by standard methods. Generally, the integral membrane protein can be obtained by lysing the membrane with detergents, such as but not limited to, sodium dodecyl sulfate (SDS), Triton X-100 polyoxyethylene ester, IpageI/nonidet P-40 (NP-40) (octylphenoxy)-polyethoxyethanol, digoxin, sodium deoxycholate, and the like, including mixtures thereof. Solubilization can be enhanced by sonication of the suspension. Soluble forms of the protein can be obtained by collecting culture fluid or by solubilizing inclusion bodies, e.g., by treatment with detergent, and, if desired, sonication or other mechanical processes, as described above. The solubilized or soluble protein can be isolated using various techniques, such as polyacrylamide gel electrophoresis (PAGE), isoelectric focusing, 2-dimensional gel electrophoresis, chromatography (e.g., ion exchange, affinity, immunoaffinity, and sizing column chromatography), centrifugation, differential solubility, immunoprecipitation, or by any other standard technique for the purification of proteins.

[0298] Alternatively, a nucleic acid or vector according to the invention can be introduced in vivo by lipofection. For the past decade, there has been increasing use of liposomes for encapsulation and transfection of nucleic acids in vitro. Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome-mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Felgner, et. al. (1987. PNAS 84/7413); Mackey, et al. (1988. Proc. Natl. Acad. Sci. USA 85 :8027-8031); Ulmer et al. (1993. Science 259 :1745-1748). The use of cationic lipids may promote encapsulation of negatively charged nucleic acids and also promote fusion with negatively charged cell membranes (Felgner and Ringold, (1989. Science 337:387-388)). Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications W095/18863 and W096/17823, and in U.S. Pat. No. 5,459,127. The use of lipofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. It is clear that directing transfection to particular cell types would be particularly preferred in a tissue with cellular heterogeneity, such as pancreas, liver, kidney, and the brain. Lipids may be chemically coupled to other molecules for the purpose of targeting [see Mackey, et. al., supra]. Targeted peptides, e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.

[0299] Other molecules also are useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., International Patent Publication WO95/21931), peptides derived from DNA binding proteins (e.g., International Patent Publication WO96/25508), or a cationic polymer (e.g., International Patent Publication WO95/21931).

[0300] It is also possible to introduce the vector in vivo as a naked DNA plasmid (see U.S. Pat. Nos. 5,693,622, 5,589,466, and 5,580,859). Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (see, Wu et al., 1992, supra; Wu and Wu, 1988, supra; Hartmut et al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990; Williams et al., 1991, Proc. Natl. Acad. Sci. USA 88:2726-2730). Receptor-mediated DNA delivery approaches can also be used (Curiel et al., 1992, Hum. Gene Ther. 3:147-154; Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432).

[0301] The term “pharmaceutically-acceptable vehicle or excipient” includes diluents and fillers which are pharmaceutically acceptable for method of administration, are sterile, and may be aqueous or oleaginous suspensions formulated using suitable dispersing or wetting agents and suspending agents. The particular pharmaceutically-acceptable carrier and the ratio of active compound to carrier are determined by the solubility and chemical properties of the composition, the particular mode of administration, and standard pharmaceutical practice.

[0302] Any nucleic acid, polypeptide, vector, or host cell of the invention will preferably be introduced in vivo in a pharmaceutically-acceptable vehicle or excipient. The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that are physiologically-tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness, and the like, when administered to a human. In general, the term “pharmaceutically-acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, for example in humans. The term “excipient” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions may be employed as excipients, for example, for injectable solutions. Suitable pharmaceutical excipients are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.

[0303] Naturally, the invention contemplates delivery of a vector that will express a therapeutically effective amount of any one of ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides for gene therapy applications. The phrase “therapeutically effective amount” is used herein to mean an amount sufficient to reduce by at least about 15 percent, at least 50 percent, at least 90 percent, or even prevent a clinically significant deficit in the activity, function, and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the host.

[0304] “Lipid profile” means the set of concentrations of cholesterol, triglyceride, lipoprotein cholesterol and other lipids in the body of a human or other animal.

[0305] An “undesirable lipid profile” is the condition in which the concentrations of cholesterol, triglyceride, or lipoprotein cholesterol are outside of the age- and gender-adjusted reference ranges. Generally, a concentration of total cholesterol>200 mg/dl, of plasma triglycerides>200 mg/dl, of LDL cholesterol>130 mg/dl, of HDL cholesterol<39 mg/dl, or a ratio of total cholesterol to HDL cholesterol>4.0 is considered to be an undesirable lipid profile. An undesirable lipid profile is associated with a variety of pathological conditions, including hyperlipidaemias, diabetes hypercholesterolaemia, arteriosclerosis, and other forms of coronary artery disease.

[0306] Nucleic Acids of the ABCA5, A6, A9, A10 Genes

[0307] Applicants have identified a novel human ABCA-like cluster of genes, designated ABCA5, ABCA6, ABCA9, and ABCA10. Applicants also have determined that the new genes are closely spaced and arranged head-to-tail in the following order ABCA5, ABCA10, ABCA6, and ABCA9 on same region of chromosome 17q24 and encode full transporters (FIG. 1).

[0308] Applicants also have determined that each new ABCA gene has a unique expression pattern, suggesting that the corresponding proteins may perform tissue-specialized functions (Example 3).

[0309] The expression patterns showed that the 6.5 kb ABCA5 transcript is almost ubiquitous, but the strongest expression was found in testis, skeletal muscle, fetal kidney and fetal liver. The ABCA9 transcript of 6 kb was found in heart and a weak signal was detected in the ovary, small intestine and testis. The ABCA6 transcript of 7 kb-long was detected with a strong signal in the liver and fetal liver, a weaker signal was detected in heart, kidney (fetal and adult), lung (fetal and adult), colon, small intestine, ovary, and testis. No signal was detected under the same conditions in the brain (fetal and adult), pancreas, placenta, skeletal muscle, leukocyte, pancreas, spleen, or thymus. The ABCA10 transcript of 6.5 kb was strongly and specifically detected in skeletal muscle and heart (FIG. 4).

[0310] Also, in situ hybridization showed the strongest ABCA9 gene expression in endothelial cells, vascular smooth muscle, and Schwann cells, and the strongest ABCA10 gene expression was identified consistently in macrophages, subsets of lymphocytes, and in Schwann cells of nerves.

[0311] Applicants have further determined transcript sequences that correspond to the full coding sequence (CDS) of the ABCA5, ABCA6, and ABCA9, and ABCA10 genes and that the ABCA6 and ABCA9 genes comprise 39 exons and 38 introns, and the ABCA10 gene comprises at least 40 exons and 39 introns. Table 1 hereinafter presents splice donors and acceptors scores (R_(i), bits) that are consistent with that of exons in other mammalian genes (Rogan et al., Hum Mutat (1998) 12, 153-171). Exons are located in exactly the same positions in all genes, although the length of some of the exons varies. Furthermore, there is a high correlation coefficient (0.990-0.997) for exon size between these genes and significant correlations (0.27-0.64) for some of the comparisons of intron sizes and R_(i) values, clearly suggesting that the genes from the 17q24 cluster arose by duplication from a common ancestor. TABLE 1 Correlations of exon size, intron size and Ri values. Exons Introns Ri-SD Ri-SA ABCA6-ABCA9 0.997 0.62 0.45 0.28 ABCA6-ABCA10 0.990 0.46 0.64 0.53 ABCA9-ABCA10 0.995 0.43 0.47 0.46

[0312] Applicants have thus characterized new exon sequences of the human ABCA6, ABCA9, and ABCA10 genes, which are particularly useful according to the invention for detecting the corresponding, ABCA6, ABCA9, and ABCA10 genes or nucleotide expression products in a sample.

[0313] Several exons of ABCA6 gene have been characterized by their nucleotide sequence and are identified in Table 2. TABLE 2 Human ABCA6 exons and intron DNA Exon or Exon start in Exon stop in Exon Exon Length Intron start Intron stop Length intron genomic genomic start in stop in of in genomic in genomic in number fragment fragment mRNA mRNA exon fragment fragment intron 1 123426 123555 1 130 130 123556 124551 996 2 124552 124692 131 271 141 124693 127799 3107 3 127800 128004 272 476 205 128005 129049 1045 4 129050 129208 477 635 159 129209 130557 1349 5 130558 130661 636 739 104 130662 131432 771 6 131433 131659 740 966 227 131660 135548 3889 7 135549 135690 967 1108 142 135691 136495 805 8 136496 136681 1109 1294 186 136682 140264 3583 9 140265 140412 1295 1442 148 140413 141892 1480 10 141893 142061 1443 1611 169 142062 147343 5282 11 147344 147402 1612 1670 59 147403 149813 2411 12 149814 149924 1671 1781 111 149925 150362 438 13 150363 150538 1782 1957 176 150539 151562 1024 14 151563 151682 1958 2077 120 151683 151939 257 15 151940 152078 2078 2216 139 152079 153026 948 16 153027 153117 2217 2307 90 153118 154359 1242 17 154360 154499 2308 2447 139 154500 157487 2988 18 157488 157604 2448 2564 117 157605 159088 1484 19 159089 159272 2565 2748 184 159273 159671 399 20 159672 159838 2749 2915 167 159839 162331 2493 21 162332 162465 2916 3049 134 162466 164365 1900 22 154366 164503 3050 3187 138 164504 167272 2769 23 167273 167380 3188 3295 108 167381 168498 1118 24 168499 168672 3296 3469 174 168673 168946 274 25 168947 169060 3470 3583 114 169061 174037 4977 26 174038 174157 3584 3703 120 174158 175757 1600 27 175758 175835 3704 3781 78 175836 177041 1206 28 177042 177133 3782 3873 92 177134 177826 693 29 177827 177947 3874 3994 121 177948 178564 617 30 178565 178682 3995 4112 118 178683 179583 901 31 179584 179675 4113 4204 92 179676 180117 442 32 180118 180272 4205 4359 155 180273 180792 520 33 180793 180868 4360 4435 76 180869 180944 76 34 180945 181039 4436 4530 95 181040 181968 929 35 181969 182088 4531 4650 120 182089 182286 198 36 182287 182427 4651 4791 141 182428 184154 1727 37 184155 184234 4792 4871 80 184235 186034 1800 38 186035 186090 4872 4927 56 186091 186225 135 39 186226 186594 4928 5296 369 186595

[0314] Thus the present invention also relates to a nucleic acid comprising any one of SEQ ID NOs: 9-47 or a complementary sequence.

[0315] The invention also relates to a nucleic acid comprising a nucleotide sequence as depicted in any one of SEQ ID NOs: 9-47 or a complementary nucleotide sequence.

[0316] The invention also relates to a nucleic acid comprising at least 8 consecutive nucleotides of any one of SEQ ID NOs: 9-47 or a complementary nucleotide sequence.

[0317] The subject of the invention is, in addition, a nucleic acid having at least 80% nucleotide identity with a nucleic acid comprising any one of SEQ ID NOs: 9-47 or a complementary nucleotide sequence.

[0318] The invention also relates to a nucleic acid having at least 85%, at least 90%, at least 95%, or at least 98% nucleotide identity with a nucleic acid comprising any one of SEQ ID NOs: 9-47.

[0319] The invention also relates to a nucleic acid hybridizing, under high stringency conditions, with a nucleic acid comprising any one of SEQ ID NOs: 9-47 or a complementary nucleotide sequence.

[0320] Several exons of the ABCA9 gene have been characterized by their nucleotide sequence and are identified in Table 3. TABLE 3 Human ABCA9 exons and intron DNA Exon or Exon start in Exon stop in Exon Exon Length Intron start Intron stop Length intron genomic genomic start in stop in of in genomic in genomic in number fragment fragment mRNA mRNA exon fragment fragment intron 1 204305 204434 1 130 130 204435 214160 9726 2 214161 214269 131 239 109 214270 215809 1540 3 215810 216017 240 447 208 216018 219963 3946 4 219964 220128 448 612 165 220129 220699 571 5 220700 220803 613 716 104 220804 221584 781 6 221585 221811 717 943 227 221812 229498 7687 7 229499 229640 944 1085 142 229641 229868 228 8 229869 230054 1086 1271 186 230055 231426 1372 9 231427 231574 1272 1419 148 231575 233023 1449 10 233024 233192 1420 1588 169 233193 236072 2880 11 236073 236131 1589 1647 59 236132 236654 523 12 236655 236765 1648 1758 111 236766 237484 719 13 237485 237660 1759 1934 176 237661 237850 190 14 237851 237970 1935 2054 120 237971 238185 215 15 238186 238324 2055 2193 139 238325 238832 508 16 238833 238923 2194 2284 91 238924 240946 2023 17 240947 241086 2285 2424 140 241087 243438 2352 18 243439 243558 2425 2544 120 243559 244713 1155 19 244714 244912 2545 2743 199 244913 246720 1808 20 246721 246887 2744 2910 167 246888 247510 623 21 247511 247644 2911 3044 134 247645 248909 1265 22 248910 249047 3045 3182 138 249048 253216 4169 23 253217 253324 3183 3290 108 253325 257064 3740 24 257065 257238 3291 3464 174 257239 257427 189 25 257428 257641 3465 3578 114 257542 269285 11744 26 269286 269405 3579 3698 120 269406 272215 2810 27 272216 272284 3699 3767 69 272285 273033 749 28 273034 273125 3768 3859 92 273126 274342 1217 29 274343 274463 3860 3980 121 274464 275369 906 30 275370 275487 3981 4098 118 275488 276181 694 31 276182 276273 4099 4190 92 276274 278975 2702 32 278976 279136 4191 4351 161 279137 280171 1035 33 280172 280247 4352 4427 76 280248 280320 73 34 280321 280415 4428 4522 95 280416 281124 709 35 281125 281244 4523 4642 120 281245 281450 206 36 281451 281591 4643 4783 141 281592 282658 1067 37 282659 282738 4784 4863 80 282739 289109 6371 38 289110 289165 4864 4919 56 289166 289286 121 39 289287 290352 4920 5981 1062 290353

[0321] Thus, the present invention also relates to a nucleic acid comprising any one of SEQ ID NOs: 48-86 or a complementary sequence.

[0322] The invention also relates to a nucleic acid comprising a nucleotide sequence as depicted in any one of SEQ ID NOs: 48-86, or a complementary nucleotide sequence.

[0323] The invention also relates to a nucleic acid comprising at least 8 consecutive nucleotides of any one of SEQ ID NOs: 48-86 or a complementary nucleotide sequence.

[0324] The subject of the invention is, in addition, a nucleic acid having at least 80% nucleotide identity with a nucleic acid comprising any one of SEQ ID NOs: 48-86 or a complementary nucleotide sequence.

[0325] The invention also relates to a nucleic acid having at least 85%, at least 90%, at least 95%, or at least 98% nucleotide identity with a nucleic acid comprising any one of SEQ ID NOs: 48-86.

[0326] The invention also relates to a nucleic acid hybridizing, under high stringency conditions, with a nucleic acid comprising any one of SEQ ID NOs: 48-86 or a complementary nucleotide sequence.

[0327] Several exons of the ABCA10 gene have been characterized by their nucleotide sequence and are identified in Table 4. TABLE 4 Human ABCA10 exons and introns DNA Exon or Exon start in Exon stop in Exon Exon Length Intron start Intron stop Length intron genomic genomic start in stop in of in genomic in genomic in number fragment fragment mRNA mRNA exon fragment fragment intron 1 20483 20769 1 287 287 20770 36437 15668 2 36438 36717 288 567 280 36718 38012 1295 3 38013 38153 568 708 141 38154 39768 1615 4 39769 39973 709 913 205 39974 42600 2627 5 42601 42765 914 1078 165 42766 43402 637 6 43403 43506 1079 1182 104 43507 45526 2020 7 45527 45753 1183 1409 227 45754 48939 3186 8 48940 49081 1410 1551 142 49082 49297 216 9 49298 49483 1552 1737 186 49484 50446 963 10 50447 50594 1738 1885 148 50595 63629 13035 11 63630 63798 1886 2054 169 63799 68175 4377 12 68176 68234 2055 2113 59 68235 70803 2569 13 70804 70914 2114 2224 111 70915 71309 395 14 71310 71485 2225 2400 176 71486 71686 201 15 71687 71806 2401 2520 120 71807 72050 244 16 72051 72189 2521 2659 139 72190 72645 456 17 72646 72736 2660 2750 91 72737 73983 1247 18 73984 74123 2751 2890 140 74124 74821 698 19 74822 74941 2891 3010 120 74942 77419 2478 20 77420 77618 3011 3209 199 77619 79655 2037 21 79656 79822 3210 3376 167 79823 82490 2668 22 82491 82624 3377 3510 134 82625 83008 384 23 83009 83146 3511 3648 138 83147 89785 6639 24 89786 89893 3649 3756 108 89894 90521 628 25 90522 90692 3757 3927 171 90693 90904 212 26 90905 91018 3928 4041 114 91019 100216 9198 27 100217 100336 4042 4161 120 100337 101145 809 28 101146 101226 4162 4242 81 101227 108376 7150 29 108377 108468 4243 4334 92 108469 109374 906 30 109375 109495 4335 4455 121 109496 110163 668 31 110164 110281 4456 4573 118 110282 110973 692 32 110974 111065 4574 4665 92 111066 111290 225 33 111291 111469 4666 4844 179 111470 111753 284 34 111754 111829 4845 4920 76 111830 111900 71 35 111901 111995 4921 5015 95 111996 112818 823 36 112819 112938 5016 5135 120 112939 113116 178 37 113117 113257 5136 5276 141 113258 115236 1979 38 115237 115316 5277 5356 80 115317 116211 895 39 116212 116267 5357 5412 56 116268 116374 107 116375 117143 5413 6181 769 117144

[0328] Thus, the invention also relates to a nucleic acid comprising any one of SEQ ID NOs: 87-126 or a complementary nucleotide sequence thereof.

[0329] The invention also relates to a nucleic acid comprising a nucleotide sequence as depicted in any one of SEQ ID NOs: 87-126 or a complementary nucleotide sequence thereof.

[0330] The invention also relates to a nucleic acid comprising at least 8 consecutive nucleotides of any one of SEQ ID NOs: 87-126 or a complementary nucleotide sequence.

[0331] The subject of the invention is, in addition, a nucleic acid having at least 80% nucleotide identity with a nucleic acid comprising any one of SEQ ID NOs: 87-126 or a complementary nucleotide sequence.

[0332] The invention also relates to a nucleic acid having at least 85%, at least 90%, at least 95%, or at least 98% nucleotide identity with a nucleic acid comprising any one of SEQ ID NOs: 87-126 or a complementary nucleotide sequence.

[0333] The invention also relates to a nucleic acid hybridizing, under high stringency conditions, with a nucleic acid comprising any one of SEQ ID NOs: 87-126 or a complementary nucleotide sequence.

[0334] cDNA Molecules Encoding Full Length ABCA5, ABCA6, ABCA9, and ABCA10 Proteins

[0335] Applicants have further determined the cDNA sequences and the full coding sequences (CDS) of the human ABCA5, A6, A9, and A10 genes, which belong to the same chromosome 17 cluster and encode full length human corresponding proteins (Example 2).

[0336] Table 5 summarizes, for each gene, the mRNA length, the coding nucleotide sequence length, and the protein size. TABLE 5 Characterization of the four ABCA on the chromosome 17 cluster mRNA Number length CDS Polyadenylation Protein of coding (bp) (bp) site (AA) exons ABCA5 6525 4929 — 1642 Nd* ABCA6 5296 4854 AATAAA 1617 38 (position 5284) ABCA9 5959 4875 — 1624 38 ABCA10 6181 4632 — 1543 37

[0337] The cDNA sequence of ABCA5 comprises 6525 nucleotides and contains a 4929 nucleotide coding sequence corresponding to a 1642 amino acid (aa) ABCA5 polypeptide produced in subjects not affected by disorders associated with cholesterol reverse transport or inflammatory lipid mediators transport. The cDNA molecule of the novel human ABCA5 gene having the nucleotide sequence as set forth in SEQ ID NO: 1 comprises an open reading frame beginning from the nucleotide at position 1011 (base A of the ATG codon for initiation of translation) to the nucleotide at position 5939 (base A of the TGA stop codon).

[0338] According to the invention, the ABCA5 cDNA comprising SEQ ID NO: 1 encodes a full length ABCA5 polypeptide of 1642 amino acids comprising the amino acid sequence of SEQ ID NO: 5.

[0339] The cDNA molecule of the novel human ABCA6 gene having the nucleotide sequence as set forth in SEQ ID NO: 2 comprises an open reading frame beginning from the nucleotide at position 176 (base A of the ATG codon for initiation of translation) to the nucleotide at position 5029 (second base A of the TAA stop codon). A polyadenylation signal (having the sequence AATAAA) is present, starting from the nucleotide at position 5284 of the sequence SEQ ID NO: 2.

[0340] According to the invention, the ABCA6 cDNA (SEQ ID NO: 2) comprises 5296 nucleotides and contains a 4854 nucleotide coding sequence that encodes a full length ABCA6 polypeptide of 1617 amino acids comprising the amino acid sequence of SEQ ID NO: 6.

[0341] The cDNA molecule of the novel human ABCA9 gene having the nucleotide sequence as set forth in SEQ ID NO: 3 comprises a coding sequence beginning from the nucleotide at position 144 (base A of the ATG codon for initiation of translation) to the nucleotide at position 5018 (second base A of the TAA stop codon).

[0342] According to the invention, the ABCA9 cDNA (SEQ ID NO: 3) comprises 5959 nucleotides and contains a 4875 nucleotide coding sequence which encodes a full length ABCA9 polypeptide of 1624 amino acids comprising the amino acid sequence of SEQ ID NO: 7.

[0343] The cDNA molecule of the novel human ABCA10 gene having the nucleotide sequence as set forth in SEQ ID NO: 4 comprises a coding sequence beginning from the nucleotide at position 880 (base A of the ATG codon for initiation of translation) to the nucleotide at position 5511 (second base A of the TAA stop codon).

[0344] According to the invention, the ABCA10 cDNA (SEQ ID NO: 4) comprises 6181 nucleotides and contains a 4632 nucleotide coding sequence which encodes a full length ABCA10 polypeptide of 1543 amino acids comprising the amino acid sequence of SEQ ID NO: 8.

[0345] The present invention is directed to a nucleic acid comprising SEQ ID NOs: 1-4 or a complementary nucleotide sequence thereof.

[0346] The invention also relates to a nucleic acid comprising a nucleotide sequence as depicted in SEQ ID NO :1-4 or a complementary nucleotide sequence thereof.

[0347] The invention also relates to a nucleic acid comprising at least eight consecutive nucleotides of SEQ ID NOS: 1-4 or a complementary nucleotide sequence thereof.

[0348] The subject of the invention is also a nucleic acid having at least 80% nucleotide identity with a nucleic acid comprising nucleotides of SEQ ID NOs: 1-4 or a nucleic acid having a complementary nucleotide sequence thereof.

[0349] The invention also relates to a nucleic acid having at least 85%, at least 90%, at least 95%, or at least 98% nucleotide identity with a nucleic acid comprising a nucleotides of SEQ ID NOs: 1-4 or a nucleic acid having a complementary nucleotide sequence thereof.

[0350] Another subject of the invention is a nucleic acid hybridizing, under high stringency conditions, with a nucleic acid comprising nucleotides of SEQ ID NOs: 1-4 or a nucleic acid having a complementary nucleotide sequence thereof.

[0351] The invention also relates to a nucleic acid encoding a polypeptide comprising an amino acid sequence of SEQ ID NOs: 5-8.

[0352] The invention relates to a nucleic acid encoding a polypeptide comprising an amino acid sequence as depicted in SEQ ID NOs: 5-8.

[0353] The invention also relates to a polypeptide comprising an amino acid sequence of SEQ ID NOs: 5-8.

[0354] The invention also relates to a polypeptide comprising anamino acid sequence as depicted in SEQ ID NOs: 5-8.

[0355] The invention also relates to a polypeptide comprising an amino acid sequence having at least 80% amino acid identity with a polypeptide comprising an amino acid sequence of SEQ ID NOs: 5-8 or a peptide fragment thereof.

[0356] The invention also relates to a polypeptide having at least 85%, at least 90%, at least 95%, or at least 98% amino acid identity with a polypeptide comprising an amino acid sequence of SEQ ID NOs: 5-8.

[0357] Preferably, a polypeptide according to the invention will have a length of 4, 5 to 10, 15, 18 or 20 to 25, 35, 40, 50, 70, 80, 100 or 200 consecutive amino acids of a polypeptide according to the invention comprising an amino acid sequence of SEQ ID NOs: 5-8.

[0358] Like the ABCA1 and ABCA4 transporters, which present 45 to 66% amino acid sequences identity, the ABCA5, ABCA6, ABCA9, and ABCA10 proteins also demonstrate high conservation as set forth in Tables 6-10 (FIG. 2). Alignment of the amino acid sequences of the ABCA5, ABCA6, ABCA9 and ABCA10 genes reveals an identity ranging from 43 to 62% along the entire sequence (Table 6). Particularly, the ABCA5, ABCA6, ABCA9, and ABCA10 proteins show 32 to 60% and 34 to 48% identity in the N-terminal (Table 7) and C-terminal (Table 8) trans-membrane domains (TMC and TMN), respectively, and 56 to 77% identity in the ATP-binding domains (NBD1 and NBD2; Tables 9 and 10). TABLE 6 Homology/Identity percentages between the amino acid sequences of ABCA5, ABCA6, ABCA8, ABCA9, ABCA10, and ABCA1 along the entire sequence Total sequence ABCA5 ABCA6 ABCA8 ABCA9 ABCA10 ABCA1 ABCA5 100/100 ABCA6 52.9/42.8 100/100 ABCA8 52.4/42.4 67/59.7 100/100 ABCA9 52.6/42.7 67.4/59.4 78.2/71.6 100/100 ABCA10 53.2/43.4 69.5/62.3 68.1/61.1 70.3/62.1 100/100 ABCA1 41.5/30.8 42.8/31 42.832 41.1/30.9 41.2/30.6 100/100

[0359] TABLE 7 Homology/Identity percentages between the amino acid sequences of ABCA5, ABCA6, ABCA9, ABCA10, ABCA1, and ABCA8 in the N terminal transmembrane domain TMN domain ABCA5 ABCA6 ABCA8 ABCA9 ABCA10 ABCA1 ABCA5 100/100 ABCA6 47/34.2 100/100 ABCA8 46.5/35 70.2/59.1 100/100 ABCA9 46.3/37.8 64.2/55.7 76.9/68.5 100/100 ABCA10 43.4/32.3 68.5/60.4 70.7/60.8 65.5/57.9 100/100 ABCA1 36.5/23.1 34.2/20 39.8/27.6 40.6/27.9 35.4/24.4 100/100

[0360] TABLE 8 Homology/Identity percentages between the amino acid sequences of ABCA5, ABCA6, ABCA9, ABCA10, ABCA1 and ABCA8 in the C terminal transmembrane domain TMC domain ABCA5 ABCA6 ABCA8 ABCA9 ABCA10 ABCA1 ABCA5 100/100 ABCA6 43.7/33.7 100/100 ABCA8 48.2/31.8 53.8/44.2 100/100 ABCA9 47.3/33.7 57.2/48.2 64.1/52.9 100/100 ABCA10 47/35.4 57/47 54.3/43 57.4/44.4 100/100 ABCA1 33/21.6 32/21.4 39/24.8 35.3/26.8 34.7/22.4 100/100

[0361] TABLE 9 Homology/Identity percentages between the amino acid sequences of ABCA5, ABCA6, ABCA9, ABCA10, ABCA1, and ABCA8 in the nucleotide Binding Domain 1 (NBD1) NBD1 domain ABCA5 ABCA6 ABCA8 ABCA9 ABCA10 ABCA1 ABCA5 100/100 ABCA6 70.2/60.3 100/100 ABCA8 71.8/62.5 85.4/78.6 100/100 ABCA9 65.5/58.2 80.2/72.8 88.5/81.8 100/100 ABCA10 69.8/62.1 83.2/77.2 82.8/79.2 81.9/75.8 100/100 ABCA1 56.8/48.5 53.5/43.5 61.2/50.5 51.7/43.8 56.5/45.6 100/100

[0362] TABLE 10 Homology/Identity percentages between the amino acid sequences of ABCA5, ABCA6, ABCA9, ABCA10, ABCA1, and ABCA8 in the nucleotide Binding Domain 2 (NBD2) NBD2 domain ABCA5 ABCA6 ABCA8 ABCA9 ABCA10 ABCA1 ABCA5 100/100 ABCA6 63/56.1 100/100 ABCA8 66.9/58.4 78/73.5 100/100 ABCA9 65.2/57.0 77.6/72.6 94.5/91.8 100/100 ABCA10 63.7/56.2 74.9/71.2 81/77.4 82.3/77.8 100/100 ABCA1 46.4/37.8 46.3/37.9 46.9/38 47.3/39 46.4/37.7 100/100

[0363] Phylogenetic analysis of the ATP-binding domains demonstrated that the N- and C-terminal domains form separate branches (FIG. 3). The C-terminal ATP-binding domains of the 17q24 genes are more closely related to the C-terminal domains of the other ABC1-like genes than to the N-terminal domains of the same proteins. Thus, the entire ABC1 subfamily appears to have arisen from a single ancestral full transporter gene. However, the genes in the 17q24 cluster form a distinct group within the ABC1 subfamily.

[0364] Nucleotide Probes and Primers

[0365] Nucleotide probes and primers hybridizing with a nucleic acid (genomic DNA, messenger RNA, cDNA) according to the invention also form part of the invention.

[0366] According to the invention, nucleic acid fragments derived from a polynucleotide comprising any one of SEQ ID NOs: 1-4 and 9-126 or of a complementary nucleotide sequence are useful for the detection of the presence of at least one copy of a nucleotide sequence of any one of the ABCA5, ABCA6, ABCA9, and ABCA10 genes or of a fragment or of a variant (containing a mutation or a polymorphism) thereof in a sample.

[0367] The nucleotide probes or primers according to the invention comprise a nucleotide sequence comprising any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence.

[0368] The nucleotide probes or primers according to the invention comprise at least 8 consecutive nucleotides of a nucleic acid comprising any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence.

[0369] Nucleotide probes or primers according to the invention may have a length of about 10, about 12, about 15, about 18 or about 20 to about 25, about 35, about 40, about 50, about 70, about 80, about 100, about 200, about 500, about 1000, or about 1500 consecutive nucleotides of a nucleic acid according to the invention, for example, a nucleic acid comprising any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence.

[0370] Alternatively, a nucleotide probe or primer according to the invention consists of and/or comprise the fragments having a length of about 12, about 15, about 18, about 20, about 25, about 35, about 40, about 50, about 100, about 200, about 500, about 1000, or about 1500 consecutive nucleotides of a nucleic acid according to the invention, for example, a nucleic acid comprising any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence.

[0371] The definition of a nucleotide probe or primer according to the invention therefore encompasses oligonucleotides that hybridize, under the high stringency hybridization conditions defined above, with a nucleic acid comprising any one of SEQ ID NOs: 1-4 and 9-126, or a complementary nucleotide sequence.

[0372] According to a preferred embodiment, a nucleotide primer according to the invention comprises a nucleotide sequence of any one of SEQ ID NOs: 127-144, or a complementary nucleic acid sequence.

[0373] Examples of primers and pairs of primers that make it possible to amplify various regions of the ABCA5 gene are presented in Table 11 below. The location of each primer of SEQ ID NOs: 127-144 within SEQ ID NO: 1 and its hybridizing region is indicated in Table 11. The abbreviation “Comp” refers to the complementary nucleic acid sequence. TABLE 11 Primers for the amplification of nucleic fragments of the ABCA5 gene Primer Located in Position in SEQ ID NO: SEQ ID NO: the sequence 127 1 3842-3860 128 1 Comp 4858-4876 129 1 Comp 4783-4801 130 1 Comp 5789-5807 131 1 5630-5648 132 1 4858-4876 133 1 Comp 3998-4016 134 1 Comp 2987-3005 135 1 Comp 3186-3208 136 1 2528-2547 137 1 Comp 3088-3107 138 1 Comp 2528-2547 139 1 Comp 845-862 140 1 789-807 141 1 Comp 1614-1633 142 1 1614-1633 143 1 Comp 537-566 144 1 Comp 202-231

[0374] According to one embodiment, a nucleotide primer according to the invention comprises a nucleotide sequence of any one of SEQ ID NOs: 145-172 or a complementary nucleic acid sequence.

[0375] Examples of primers and pairs of primers that make it possible to amplify various regions of the ABCA6 gene are presented in Table 12 below. The location of each primer of SEQ ID NOs: 145-172 within SEQ ID NO: 2 and its hybridizing region is indicated in Table 12. The abbreviation “Comp” refers to the complementary nucleic acid sequence. TABLE 12 Primers for the amplification of nucleic fragments of the ABCA6 gene Primer Located in Position in Region SEQ ID NO: SEQ ID NO: the sequence for hybridization 145 2 202-221 Exon 2 146 2 Comp 435-461 Exon 3 147 2 Comp 645-672 Exon 5 148 2 637-656 Exon 5 149 2 754-772 Exon 6 150 2 Comp 758-778 Exon 6 151 2 Comp 773-792 Exon 6 152 2 1288-1307 Exon 8-9 153 2 1321-1341 Exon 9 154 2 Comp 1322-1343 Exon 9 155 2 Comp 1592-1574 Exon 10 156 2 1761-1782 Exon 12 157 2 Comp 1928-1949 Exon 13 158 2 1944-1968 Exon 13-14 159 2 Comp 2041-2061 Exon 14 160 2 Comp 2371-2392 Exon 17 161 2 2350-2371 Exon 17 162 2 2806-2884 Exon 20 163 2 Comp 2884-2902 Exon 20 164 2 3292-3313 Exon 23-24 165 2 Comp 3357-3339 Exon 24 166 2 Comp 3746-3767 Exon 27 167 2 3754-3775 Exon 27 168 2 4176-4194 Exon 31 169 2 Comp 4248-4194 Exon 31-32 170 2 4743-4763 Exon 36 171 2 Comp 4796-4778 Exon 36-37 172 2 Comp 5262-5244 Exon 39

[0376] According another embodiment, a nucleotide primer according to the invention comprises a nucleotide sequence of any one of SEQ ID NOs: 173-203 or a complementary nucleic acid sequence.

[0377] Examples of primers and pairs of primers that make it possible to amplify various regions of the ABCA9 gene are presented in Table 13 below. The location of each primer of SEQ ID NOs: 173-203 within SEQ ID NO: 3 and its hybridizing region is indicated in Table 13. The abbreviation “Comp” refers to the complementary nucleic acid sequence. TABLE 13 Primers for the amplification of nucleic fragments of the ABCA9 gene Primer Located in Position in Region SEQ ID NO: SEQ ID NO: the sequence for hybridization 173 3 160-178 Exon 2 174 3 Comp 789-808 Exon 6 175 3 786-804 Exon 6 176 3 Comp 1434-1455 Exon 10 177 3 1305-1323 Exon 9 178 3 Comp 1632-1653 Exon 11-12 179 3 1495-1516 Exon 10 180 3 1866-1887 Exon 13 181 3 Comp 1905-1923 Exon 13 182 3 Comp 2349-2368 Exon 17 183 3 2253-2272 Exon 17 184 3 Comp 2822-2843 Exon 20 185 3 2645-2663 Exon 19 186 3 Comp 3089-3110 Exon 22 187 3 3240-3260 Exon 23 188 3 3023-3044 Exon 21 189 3 Comp 3801-3820 Exon 28 190 3 Comp 3377-3398 Exon 24 191 3 3626-3646 Exon 26 192 3 Comp 4191-4209 Exon 32 193 3 3964-3984 Exon 29-30 194 3 Comp 4784-4803 Exon 37 195 3 5230-5247 Exon 39 196 3 4694-4715 Exon 36 197 3 Comp 4977-4994 Exon 39 198 3 5541-5561 Exon 39 199 3 Comp 5960-5981 Exon 39 200 3 Comp 5541-5562 Exon 39 201 3 24-45 Exon 1 202 3 Comp 384-408 Exon 3 203 3 Comp 311-337 Exon 3

[0378] According to another embodiment, a nucleotide primer according to invention comprises a nucleotide sequence of any one of SEQ ID NOs: 204-217 complementary nucleic acid sequence thereof.

[0379] Examples of primers and pairs of primers that make it possible to amplify various regions of the ABCA10 gene are presented in Table 14 below. The location of each primer of SEQ ID NOs: 204-217 within SEQ ID NO: 4 and its hybridizing region is indicated in Table 14. The abbreviation “Comp” refers to the complementary nucleic acid sequence. TABLE 14 Primers for the amplification of nucleic fragments of the ABCA10 gene Primer Located in Position in Region SEQ ID NO: SEQ ID NO: the sequence for hybridization 204 4 1421-1440 Exon 8 205 4 Comp 1610-1629 Exon 9 206 4 2417-2434 Exon 15 207 4 Comp 2605-2623 Exon 16 208 4 Comp 3737-3754 Exon 24 209 4 Comp 814-839 Exon 4 210 4 Comp 733-757 Exon 4 211 4 61-86 Exon 1 212 4 628-643 Exon 3 213 4 3564-3583 Exon 23 214 4 Comp 4450-4468 Exon 30-31 215 4 Comp 5442-5459 Exon 40 216 4 3050-3070 Exon 20 217 4 Comp 4848-4866 Exon 34

[0380] According to another embodiment, probes and primers according to invention comprise all or part of a nucleotide sequence comprising any one of SEQ ID NOs: 127-217 or a nucleic acid having a complementary nucleic acid sequence.

[0381] A nucleotide primer or probe according to the invention may be prepared by any suitable method well known to persons skilled in the art, including by cloning and action of restriction enzymes or by direct chemical synthesis according to techniques such as the phosphodiester method by Narang et al. (1979, Methods Enzymol, 68:90-98) or by Brown et al. (1979, Methods Enzymol, 68:109-151), the diethylphosphoramidite method by Beaucage et al. (1981, Tetrahedron Lett, 22: 1859-1862), or the technique on a solid support described in EU patent No. EP 0,707,592.

[0382] Each of the nucleic acids according to the invention, including the oligonucleotide probes and primers described above, may be labeled, if desired, by incorporating a marker which can be detected by spectroscopic, photochemical, biochemical, immunochemical or chemical means. For example, such markers may consist of radioactive isotopes (³²P, ³³P, ³H, ³⁵S), fluorescent molecules (5-bromodeoxyuridine, fluorescein, acetylaminofluorene, digoxigenin) or ligands such as biotin. The labeling of the probes may be carried out by incorporating labeled molecules into the polynucleotides by primer extension or, alternatively, by addition to the 5′ or 3′ ends. Examples of nonradioactive labeling of nucleic acid fragments are described in particular in French patent No. 78 109 75 or in the articles by Urdea et al. (1988, Nucleic Acids Research, 11:4937-4957) or Sanchez-pescador et al. (1988, J. Clin. Microbiol., 26(10):1934-1938).

[0383] The nucleotide probes and primers according to the invention may have structural characteristics of the type to allow amplification of the signal, such as the probes described by Urdea et al. (1991, Nucleic Acids Symp Ser., 24:197-200) or alternatively in European patent No. EP-0,225,807 (CHIRON).

[0384] The oligonucleotide probes according to the invention may be used, for example, in Southern-type hybridizations with genomic DNA or, alternatively, in northern-type hybridizations with the corresponding messenger RNA when the expression of the corresponding transcript is sought in a sample.

[0385] The probes and primers according to the invention may also be used for the detection of products of PCR amplification or, alternatively, for the detection of mismatches.

[0386] Nucleotide probes or primers according to the invention may be immobilized on a solid support. Such solid supports are well known to persons skilled in the art and comprise surfaces of wells of microtiter plates, polystyrene beads, magnetic beads, nitrocellulose bands, or microparticles such as latex particles.

[0387] Consequently, the present invention also relates to a method of detecting the presence of a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126 or of a complementary nucleotide sequence, or a nucleic acid fragment or variant of any one of SEQ ID NOs: 1-4 and 9-126 or of a complementary nucleotide sequence in a sample, said method comprising:

[0388] 1) bringing one or more nucleotide probes or primers according to the invention into contact with the sample to be tested;

[0389] 2) detecting the complex that may have formed between the probe(s) and the nucleic acid present in the sample.

[0390] According to one embodiment of the method of detection according to the invention, the oligonucleotide probes and primers are immobilized on a support.

[0391] According to another aspect, the oligonucleotide probes and primers comprise a detectable marker.

[0392] The invention relates, in addition, to a box or kit for detecting the presence of a nucleic acid according to the invention in a sample, said box or kit comprising:

[0393] a) one or more nucleotide probe(s) or primer(s) as described above;

[0394] b) where appropriate, the reagents necessary for the hybridization reaction.

[0395] According to one aspect, the detection box or kit is characterized in that the probe(s) or primer(s) are immobilized on a support.

[0396] According to another aspect, the detection box or kit is characterized in that the oligonucleotide probes comprise a detectable marker.

[0397] According to another embodiment of the detection kit described above, such a kit comprises a plurality of oligonucleotide probes and/or primers in accordance with the invention that may be used to detect a target nucleic acid of interest or, alternatively, to detect mutations in the coding regions and/or in the non-coding regions of the nucleic acids according to the invention, for example, of nucleic acids comprising any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence.

[0398] Thus, the probes according to the invention immobilized on a support may be ordered into matrices such as “DNA chips”. Such ordered matrices have been described in U.S. Pat. No. 5,143,854 and in published PCT applications WO 90/15070 and WO 92/10092.

[0399] Support matrices on which oligonucleotide probes have been immobilized at a high density are, for example, described in U.S. Pat. No. 5,412,087 and in published PCT application WO 95/11995.

[0400] The nucleotide primers according to the invention may be used to amplify any one of the nucleic acids according to the invention, for example, a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126 or of a complementary nucleotide sequence. Alternatively, the nucleotide primers according to the invention may be used to amplify a nucleic acid fragment or variant of any one of SEQ ID NOs: 1-4 and 9-126 or of a complementary nucleotide sequence.

[0401] In one embodiment, the nucleotide primers according to the invention may be used to amplify a nucleic acid comprising any one of SEQ ID NOs: 1-4 and 9-126, or as depicted in any one of SEQ ID NOs: 1-4 and 9-126, or of a complementary nucleotide sequence.

[0402] Another subject of the invention relates to a method for amplifying a nucleic acid according to the invention, for example, a nucleic acid comprising a) any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence or b) as depicted in any one of SEQ ID NOs:1-4 and 9-126 or of a complementary nucleotide sequence, contained in a sample, said method comprising:

[0403] a) bringing the sample in which the presence of the target nucleic acid is suspected into contact with a pair of nucleotide primers whose hybridization position is located, respectively, on the 5′ side and on the 3′ side of the region of the target nucleic acid whose amplification is sought, in the presence of the reagents necessary for the amplification reaction;

[0404] b) performing an amplification reaction; and

[0405] c) detecting the amplified nucleic acids.

[0406] To carry out the amplification method as defined above, use may be made of any of the nucleotide primers described above.

[0407] The subject of the invention is, in addition, a box or kit for amplifying a nucleic acid according to the invention, for example, a nucleic acid comprising any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence, or as depicted in any one of SEQ ID NOs: 1-4 and 9-126 or of a complementary nucleotide sequence, said box or kit comprising:

[0408] a) a pair of nucleotide primers in accordance with the invention, whose hybridization position is located, respectively, on the 5′ side and 3′ side of the target nucleic acid whose amplification is sought; and optionally,

[0409] b) reagents necessary for the amplification reaction.

[0410] Such an amplification box or kit may comprise at least one pair of nucleotide primers as described above.

[0411] The subject of the invention is, in addition, a box or kit for amplifying all or part of a nucleic acid comprising any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence, said box or kit comprising:

[0412] 1) a pair of nucleotide primers in accordance with the invention, whose hybridization position is located, respectively, on the 5′ side and 3′ side of the target nucleic acid whose amplification is sought; and optionally,

[0413] 2) reagents necessary for an amplification reaction.

[0414] Such an amplification box or kit may comprise at least one pair of nucleotide primers as described above.

[0415] The invention also relates to a box or kit for detecting the presence of a nucleic acid according to the invention in a sample, said box or kit comprising:

[0416] a) one or more nucleotide probes according to the invention;

[0417] b) where appropriate, reagents necessary for a hybridization reaction.

[0418] According to one embodiment, the detection box or kit is characterized in that the nucleotide probe(s) and primer(s)are immobilized on a support.

[0419] According to another embodiment, the detection box or kit is characterized in that the nucleotide probe(s) and primer(s) comprise a detectable marker.

[0420] According to another embodiment of the detection kit described above, such a kit will comprise a plurality of oligonucleotide probes and/or primers in accordance with the invention that may be used to detect target nucleic acids of interest or, alternatively, to detect mutations in the coding regions and/or the non-coding regions of the nucleic acids according to the invention. The target nucleic acid may comprise a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126 or of a complementary nucleic acid sequence. Alternatively, the target nucleic acid may be a nucleic acid fragment or variant of a nucleic acid comprising any one of SEQ ID NOs: 1-4 and 9-126 or of a complementary nucleotide sequence.

[0421] According to the present invention, a primer according to the invention comprises, generally, all or part of any one of SEQ ID NOs: 127-217 or a complementary sequence.

[0422] The nucleotide primers according to the invention are particularly useful in methods of genotyping subjects and/or of genotyping populations, in particular in the context of studies of association between particular allele forms or particular forms of groups of alleles (haplotypes) in subjects and the existence of a particular phenotype (character) in these subjects, for example, the predisposition of these subjects to develop diseases linked to a deficiency of cholesterol reverse transport and inflammation signaling lipids or, alternatively, the predisposition of these subjects to develop a pathology whose candidate chromosomal region is situated on chromosome 17, more precisely on the 17q arm and, still more precisely, in the 17q24 locus.

[0423] Recombinant Vectors

[0424] The invention also relates to a recombinant vector comprising a nucleic acid according to the invention. “Vector” for the purposes of the present invention will be understood to mean a circular or linear DNA or RNA molecule that is either in single-stranded or double-stranded form.

[0425] A recombinant vector may comprise a nucleic acid chosen from the following nucleic acids:

[0426] a) a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126 or of a complementary nucleotide sequence,

[0427] b) a nucleic acid comprising a nucleotide sequence as depicted in any one of SEQ ID NOs: 1-4 and 9-126 or of a complementary nucleotide sequence,

[0428] c) a nucleic acid having at least eight consecutive nucleotides of a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126 or of a complementary nucleotide sequence;

[0429] d) a nucleic acid having at least 80% nucleotide identity with a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence;

[0430] e) a nucleic acid having at least 85%, at least 90%, at least 95%, or at least 98% nucleotide identity with a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence;

[0431] f) a nucleic acid hybridizing, under high stringency hybridization conditions, with a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence;

[0432] g) a nucleic acid encoding a polypeptide comprising an amino acid sequence of SEQ ID NOs: 5-8; and

[0433] h) a nucleic acid encoding a polypeptide comprising amino acid sequence selected from SEQ ID NOs: 5-8.

[0434] According to one embodiment, a recombinant vector according to the invention is used to amplify a nucleic acid inserted therein, following transformation or transfection of a desired cellular host.

[0435] According to another embodiment, a recombinant vector according to the invention corresponds to an expression vector comprising, in addition to a nucleic acid in accordance with the invention, a regulatory signal or nucleotide sequence that directs or controls transcription and/or translation of the nucleic acid and its encoded mRNA.

[0436] According to another embodiment, a recombinant vector according to the invention may comprise the following components:

[0437] (1) an element or signal for regulating the expression of the nucleic acid to be inserted, such as a promoter and/or enhancer sequence;

[0438] (2) a nucleotide coding region comprised within the nucleic acid in accordance with the invention to be inserted into such a vector, said coding region being placed in phase with the regulatory element or signal described in (1); and

[0439] (3) an appropriate nucleic acid for initiation and termination of transcription of the nucleotide coding region of the nucleic acid described in (2).

[0440] In addition, the recombinant vectors according to the invention may include one or more origins for replication in the cellular hosts in which their amplification or their expression is sought, markers or selectable markers.

[0441] By way of example, the bacterial promoters may be the LacI or LacZ promoters, the T3 or T7 bacteriophage RNA polymerase promoters, the lambda phage PR or PL promoters.

[0442] The promoters for eukaryotic cells comprise the herpes simplex virus (HSV) virus thymidine kinase promoter or, alternatively, the mouse metallothionein-L promoter.

[0443] Generally, for the choice of a suitable promoter, persons skilled in the art can refer to the book by Sambrook et al. (1989, Molecular cloning: a laboratory manual. 2ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) cited above or to the techniques described by Fuller et al. (1996, Immunology, In: Current Protocols in Molecular Biology, Ausubel et al.(eds.).

[0444] When the expression of the genomic sequence of any one of the ABCA5, ABCA6, ABCA9, and ABCA10 genes is sought, use may be made of the vectors capable of containing large insertion sequences. In one embodiment, bacteriophage vectors like the P1 bacteriophage vectors, such as the vector p158 or the vector p158/neo8 described by Sternberg (1992, Trends Genet., 8:1-16; 1994, Mamm. Genome, 5:397-404), will be used.

[0445] The bacterial vectors according to the invention include, for example, the vectors pBR322(ATCC37017) or, alternatively, vectors such as pAA223-3 (Pharmacia, Uppsala, Sweden), and pGEM1 (Promega Biotech, Madison, Wis., United States).

[0446] There may also be cited other commercially available vectors, such as the vectors pQE70, pQE60, pQE9 (Qiagen), psiX174, pBluescript SA, pNH8A, pNH16A, pNH18A, pNH46A, pWLNEO, pSV2CAT, pOG44, pXTI, and pSG (Stratagene).

[0447] The invention also encompasses vectors of the baculovirus type such as the vector pVL1392/1393 (Pharmingen), which is used to transfect cells of the Sf9 line (ATCC No. CRL 1711) derived from Spodoptera frugiperda.

[0448] Vectors according to the invention may also be adenoviral vectors, such as the human adenovirus of type 2 or 5.

[0449] A recombinant vector according to the invention may also be a retroviral vector or an adeno-associated vector (AAV). Adeno-associated vectors are, for example, described by Flotte et al. (1992, Am. J. Respir. Cell Mol. Biol., 7:349-356), Samulski et al. (1989, J. Virol., 63:3822-3828), or McLaughlin B A et al. (1996, Am. J. Hum. Genet., 59:561-569).

[0450] To allow the expression of a polynucleotide according to the invention, the polynucleotide must be introduced into a host cell. The introduction of a polynucleotide according to the invention into a host cell may be carried out in vitro, according to techniques well known to persons skilled in the art for transforming or transfecting cells, either in primary culture or in the form of cell lines. It is also possible to carry out the introduction of a polynucleotide according to the invention in vivo or ex vivo, for the prevention or treatment of diseases linked to ABC A5, A6, A9 or A10 deficiencies.

[0451] To introduce a polynucleotide or vector of the invention into a host cell, a person skilled in the art can use various techniques, such as calcium phosphate coprecipitation (Graham et al., 1973, Virology, 52:456-457 ; Chen et al., 1987, Mol. Cell. Biol., 7: 2745-2752), DEAE Dextran (Gopal, 1985, Mol. Cell. Biol., 5:1188-1190), electroporation (Tur-Kaspa, 1896, Mol. Cell. Biol., 6:716-718 ; Potter et al., 1984, Proc Natl Acad Sci U S A., 81(22):7161-5), direct microinjection (Harland et al., 1985, J. Cell. Biol., 101:1094-1095), liposomes charged with DNA (Nicolau et al., 1982, Methods Enzymol., 149:157-76; Fraley et al., 1979, Proc. Natl. Acad. Sci. USA, 76:3348-3352).

[0452] Once the polynucleotide has been introduced into the host cell, it may be stably integrated into the genome of the cell. The intergration may be achieved at a precise site of the genome, by homologous recombination, or it may be random. In some embodiments, the polynucleotide may be stably maintained in the host cell in the form of an episome fragment, the episome comprising sequences allowing the retention and the replication of the latter, either independently or in a synchronized manner with the cell cycle.

[0453] According to a specific embodiment, a method of introducing a polynucleotide according to the invention into a host cell, for example, a host cell obtained from a mammal in vivo, comprises a step during which a preparation comprising a pharmaceutically-compatible vector and a “naked” polynucleotide according to the invention, placed under the control of appropriate regulatory sequences, is introduced by local injection at the site of the chosen tissue, for example, myocardial tissue, the “naked” polynucleotide being absorbed by the myocytes of this tissue.

[0454] Compositions for use in vitro and in vivo comprising “naked” polynucleotides are, for example, described in PCT Application No. WO 95/11307 (Institut Pasteur, Inserm, University of Ottawa), as well as in the articles by Tacson et al. (1996, Nature Medicine, 2(8):888-892) and Huygen et al. (1996, Nature Medicine, 2(8):893-898).

[0455] According to a specific embodiment of the invention, a composition is provided for the in vivo production of any one of ABCA5, ABCA6, ABCA9, and ABCA10 proteins. This composition comprises a polynucleotide encoding the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides placed under the control of appropriate regulatory sequences in solution in a physiologically-acceptable vector.

[0456] The quantity of vector that is injected into the host organism chosen varies according to the site of the injection. As a guide, there may be injected between about 0.1 and about 100 μg of polynucleotide encoding the ABCA5, ABCA6, ABCA9, and ABCA10 proteins into the body of an animal, for example, into a subject likely to develop a disease linked to ABCA5, A6, A9, or A10 deficiencies.

[0457] Consequently, the invention also relates to a composition intended for the prevention of or treatment of a patient or subject affected by ABCA5, A6, A9, or A10 deficiencies, comprising a nucleic acid encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins in combination with one or more physiologically-compatible excipients.

[0458] Such a composition may comprise a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOs: 1-4, wherein the nucleic acid is placed under the control of an appropriate regulatory element or signal.

[0459] The subject of the invention is, in addition, a composition intended for the prevention of or treatment of a patient or a subject affected by an ABCA5, A6, A9 or A10 deficiency, comprising a recombinant vector according to the invention in combination with one or more physiologically-compatible excipients.

[0460] The invention also relates to the use of a nucleic acid according to the invention encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins for the manufacture of a medicament intended for the prevention of atherosclerosis in various forms or for the treatment of subjects affected by a dysfunction of cholesterol reverse transport or inflammatory liphophilic substances transport.

[0461] The invention also relates to the use of a recombinant vector according to the invention comprising a nucleic acid encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins for the manufacture of a medicament intended for the prevention of atherosclerosis in various forms or more particularly for the treatment of subjects affected by a dysfunction of cholesterol reverse transport or inflammatory liphophilic substances transport.

[0462] The subject of the invention is therefore also a recombinant vector comprising a nucleic acid according to the invention that encodes any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins or polypeptides.

[0463] The invention also relates to the use of such a recombinant vector for the preparation of a pharmaceutical composition intended for the treatment and/or for the prevention of diseases or conditions associated with a deficiency of cholesterol reverse transport or inflammatory lipophilic substances transport.

[0464] The present invention also relates to the use of cells genetically modified ex vivo with a recombinant vector according to the invention and to cells producing a recombinant vector, wherein the cells are implanted in the body to allow a prolonged and effective expression in vivo of at least a biologically active ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides.

[0465] Vectors Useful in Methods of Somatic Gene Therapy and Composition Containing Such Vectors

[0466] The present invention also relates to a new therapeutic approach for the treatment of pathologies linked to ABCA5, A6, A9, or A10 deficiencies. It provides an advantageous solution to the disadvantages of the prior art by demonstrating the possibility of treating the pathologies of ABCA5, A6, A9, or A10 deficiencies by gene therapy by the transfer and expression in vivo of a gene encoding at least one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins involved in the transport of lipophilic substances. The invention offers a simple means allowing a specific and effective treatment of related pathologies such as, for example, atherosclerosis, inflammation, cardiovascular diseases, metabolic diseases, and lipophilic substance-related pathologies.

[0467] Gene therapy consists in correcting a deficiency or an abnormality (mutation, aberrant expression, and the like) and in bringing about the expression of a protein of therapeutic interest by introducing genetic information into the affected cell or organ. This genetic information may be introduced either ex vivo into a cell extracted from the organ, the modified cell then being reintroduced into the body, or directly in vivo into the appropriate tissue. In this second case, various techniques exist, among which various transfection techniques involving complexes of DNA and DEAE-dextran (Pagano et al. (1967. J. Virol., 1:891), of DNA and nuclear proteins (Kaneda et al., 1989, Science 243:375), of DNA and lipids (Felgner et al., 1987, PNAS 84:7413), the use of liposomes (Fraley et al., 1980, J.Biol.Chem., 255:10431), and the like. More recently, the use of viruses as vectors for the transfer of genes has appeared as a promising alternative to these physical transfection techniques. In this regard, various viruses have been tested for their ability to infect certain cell populations. In particular, the retroviruses (RSV, HMS, MMS, and the like), the herpes simpex viruses (HSV), the adeno-associated viruses, and the adenoviruses may be mentioned.

[0468] The present invention therefore also relates to a new therapeutic approach to the treatment of pathologies linked to ABCA5, A6, A9, or A10 deficiencies, which consists of transferring and expressing in vivo genes encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides. For example, Applicants have now found that it is possible to construct recombinant vectors comprising a nucleic acid encoding at least one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins, to administer these recombinant vectors in vivo, and that this administration allows a stable and effective expression of at least one of the biologically active ABCA5, ABCA6, ABCA9, and ABCA10 proteins in vivo, with no cytopathological effect.

[0469] Adenoviruses are efficient vectors for the transfer and the expression of any one of the ABCA5, ABCA6, ABCA9, and ABCA10 genes. The use of recombinant adenoviruses as vectors makes it possible to obtain sufficiently high levels of expression of these genes to produce the desired therapeutic effect. The use of other viral vectors such as retroviruses or adeno-associated viruses (MV) that allow a stable expression of the gene is part of the invention.

[0470] The present invention is thus likely to offer a new approach to the treatment and prevention of ABCA5, A6, A9, and A10 deficiencies.

[0471] The subject of the invention is therefore also a defective recombinant virus comprising a nucleic acid according to the invention that encodes at least one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins or polypeptides involved in the metabolism of lipophilic substances.

[0472] The invention also relates to the use of such a defective recombinant virus for the preparation of a composition which may be useful for the treatment and/or for the prevention of ABCA5, A6, A9 or A10 deficiencies.

[0473] The present invention also relates to the use of cells genetically modified ex vivo with such a defective recombinant virus according to the invention, and to cells producing a defective recombinant virus, wherein the cells are implanted in the body, to allow a prolonged and effective expression in vivo of at least one biologically active ABCA5, ABCA6, ABCA9, or ABCA10 polypeptide.

[0474] The present invention is particularly advantageous because it is possible to induce a controlled expression, without harmful effect, of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins in organs that are not normally involved in the expression of those proteins. In particular, a significant release of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins is obtained by implantation of cells producing vectors of the invention or infected ex vivo with vectors of the invention.

[0475] The activity of these ABC protein transporters produced in the context of the present invention may be of the human or animal ABCA5, ABCA6, ABCA9, and ABCA10 type. The nucleotide sequence used in the context of the present invention may be a cDNA, a genomic DNA (gDNA), an RNA (in the case of retroviruses), or a hybrid construct consisting, for example, of a cDNA into which one or more introns (gDNA) has been inserted. It may also involve synthetic or semisynthetic sequences. In one embodiment of the invention, a cDNA or a gDNA is used. The use of a gDNA allows for better expression in human cells.

[0476] To allow their incorporation into a viral vector according to the invention, these nucleotide sequences may be modified, for example, by site-directed mutagenesis, for example, for the insertion of appropriate restriction sites. In the context of the present invention, the use of a nucleic sequence encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins is contemplated. Moreover, it is possible to use a construct encoding a derivative of any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins. A derivative of any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins comprises, for example, any sequence obtained by mutation, deletion, and/or addition relative to the native sequence and encoding a product retaining the lipophilic subtances transport activity. These modifications may be made by techniques known to a person skilled in the art (see general molecular biological techniques below). The biological activity of the derivatives thus obtained can then be easily determined, as indicated in the examples of the measurement of the efflux of the substrate from cells. The derivatives for the purposes of the invention may also be obtained by hybridization from nucleic acid libraries using as a probe the native sequence or a fragment thereof. These derivatives are, for example, molecules having a higher affinity for their binding sites, molecules exhibiting greater resistance to proteases, molecules having a higher therapeutic efficacy or fewer side effects, or optionally new biological properties. The derivatives also include the modified DNA sequences allowing improved expression in vivo.

[0477] In one embodiment, the present invention relates to a defective recombinant virus comprising a cDNA encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides. In another embodiment of the invention, a defective recombinant virus comprises a genomic DNA (gDNA) encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides. The ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides may comprise an amino acid sequence selected from SEQ ID NOs: 5-8, respectively.

[0478] The vectors of the invention may be prepared from various types of viruses. For example, vectors derived from adenoviruses, adeno-associated viruses (MV), herpesviruses (HSV) or retroviruses may be used. An adenovirus may be used for direct administration or for the ex vivo modification of cells intended to be implanted. Alternatively, a retrovirus may be used for the implantation of producing cells.

[0479] The viruses according to the invention are usually defective, that is to say that they are incapable of autonomously replicating in the target cell. Generally, the genome of the defective viruses used in the context of the present invention lacks at least the sequences necessary for the replication of said virus in the infected cell. These regions may be either eliminated (completely or partially), made nonfunctional, or substituted with other sequences, for example, with the nucleic sequence encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins. The defective virus may retain, however, the sequences of its genome that are necessary for the encapsidation of the viral particles.

[0480] With regard to adenoviruses, various serotypes, whose structure and properties vary somewhat, have been characterized, for example, human adenoviruses of type 2 or 5 (Ad 2 or Ad 5) and adenoviruses of animal origin (see Application WO 94/26914). Among the adenoviruses of animal origin that can be used in the context of the present invention, there may be mentioned adenoviruses of canine, bovine, murine (example: Mav1, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian or simian (example: SAV) origin. Preferably, the adenovirus of animal origin is a canine adenovirus, for example, a CAV2 adenovirus [Manhattan or A26/61 strain (ATCC VR-800) for example]. Adenoviruses of human or canine or mixed origin may be used in the context of the invention. In general, the defective adenoviruses of the invention comprise the ITRs, a sequence allowing encapsidation, and a sequence encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins. In general, in the genome of the adenoviruses of the invention, the at least E1 region is made nonfunctional. In addition, in the genome of the adenoviruses of the invention, at least one of the E2, E4 and LI -L5 genes may also be nonfunctional. These viral genes may be made nonfunctional by any technique known to a person skilled in the art, for example, by total suppression, by substitution, by partial deletion, or by addition of one or more bases in the inactivated gene(s). Such modifications may be obtained in vitro (on the isolated DNA) or in situ, for example, by means of genetic engineering techniques or by treatment with mutagenic agents. Other regions of the virus also may be modified, for example, the E3 (WO95/02697), E2 (WO94/28938), E4 (WO94/28152, WO94/12649, WO95/02697) and L5 (WO95/02697) regions. According to one embodiment, the adenovirus according to the invention comprises a deletion in the E1 and E4 regions and the sequence encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins is inserted at the site of the inactivated E1 region. According to another embodiment, the virus comprises a deletion in the E1 region at the site of which the E4 region and the sequence encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins (French Patent Application FR94 13355) is inserted.

[0481] The defective recombinant adenoviruses according to the invention may be prepared by any technique known to persons skilled in the art (Levrero et al., 1991 Gene 101; EP 185 573; and Graham, 1984, EMBO J., 3:2917). For example, they may be prepared by homologous recombination between an adenovirus and a plasmid carrying, inter alia, the nucleic acid encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins. The homologous recombination occurs after cotransfection of said adenoviruses and plasmid into an appropriate cell line. The cell line used must (i) be transformable by said elements and (ii) contain the sequences required to complement the part of the defective adenovirus genome, which may be in integrated form in order to avoid the risks of recombination. By way of example of a line, there may be mentioned the human embryonic kidney line 293 (Graham et al., 1977, J. Gen. Virol., 36:59), which contains the left part of the genome of an Ad5 adenovirus (12%) integrated into its genome or lines capable of complementing the E1 and E4 functions as described in particular in Application Nos. WO 94/26914 and WO95/02697.

[0482] The adeno-associated viruses (AAV) are DNA viruses of relatively small size, which integrate into the genome of the cells that they infect in a stable and site-specific manner. AAVs are capable of infecting a broad spectrum of cells without causing any effect on cellular growth, morphology or differentiation. Moreover, AAVs do not appear to be involved in pathologies in humans. The genome of AAVs has been cloned, sequenced, and characterized. It comprises about 4700 bases and contains an inverted repeat region (ITR) of about 145 bases at each end, which serves as the replication origin for the virus. The remainder of the genome is divided into 2 essential regions carrying the encapsidation functions: the left hand part of the genome, which contains the rep gene involved in the viral replication and the expression of the viral genes; the right hand part of the genome, which contains the cap gene encoding the virus capsid proteins.

[0483] The use of vectors derived from AAVs for the transfer of genes in vitro and in vivo has been described in the literature (see in particular WO 91/18088; WO 93/09239; U.S. Pat. Nos. 4,797,368, 5,139,941, EP 488 528). These applications describe various constructs derived from AAVs in which the rep and/or cap genes are deleted and replaced by a gene of interest and their use for transferring in vitro (cells in culture) or in vivo (directly into an organism) a gene of interest. However, none of these documents either describes or suggests the use of a recombinant MV for the transfer and expression in vivo or ex vivo of any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins or the advantages of such a transfer. The defective recombinant AAVs according to the invention may be prepared by cotransfection, into a cell line infected with a human helper virus (for example, an adenovirus), of a plasmid containing the sequence encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins bordered by two MV inverted repeat regions (ITR) and of a plasmid carrying the MV encapsidation genes (rep and cap genes). The recombinant MVs produced are then purified by conventional techniques.

[0484] The construction of recombinant herpesvirus and retrovirus vectors has been widely described in the literature, for example, in Breakfield et al., (1991, New Biologist, 3:203); EP 453242, EP178220, Bernstein et al. (1985); McCormick, (1985. BioTechnology, 3:689), and the like.

[0485] Retroviruses are integrating viruses, which infect dividing cells. The genome of the retroviruses comprises two long terminal repeats (LTRs), an encapsidation sequence, and three protein coding regions (gag, pol, and env). In the recombinant vectors derived from retroviruses, the gag, pol, and env genes are generally deleted, completely or partially, and replaced with a heterologous nucleic acid sequence of interest. These vectors may be produced from various types of retroviruses such as, for example, MoMuLV (“murine moloney leukemia virus”; also called MoMLV), MSV (“murine moloney sarcoma virus”), HaSV (“harvey sarcoma virus”); SNV (“spleen necrosis virus”); RSV (“rous sarcoma virus”) or Friend's virus.

[0486] To construct recombinant retroviruses containing a sequence encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins according to the invention, a plasmid containing, for example, the LTRs, the encapsidation sequence, and the coding sequence is usually constructed and used to transfect a so-called encapsidation cell line, which is capable of providing in trans the retroviral functions deficient in the plasmid. Generally, the encapsidation lines are capable of expressing the gag, pol, and env genes. Such encapsidation lines have been described in the prior art, for example, the PA317 line (U.S. Pat. No. 4,861,719), the PsiCRIP line (WO 90/02806), and the GP+envAm-12 line (WO 89/07150). Moreover, the recombinant retroviruses may contain modifications at the level of the LTRs in order to suppress transcriptional activity, as well as extended encapsidation sequences, containing a portion of the gag gene (Bender et al., 1987, J. Virol., 61:1639). The recombinant retroviruses produced are then purified by conventional techniques.

[0487] In one embodiment of the invention, a defective recombinant adenovirus is used. The advantageous properties of adenoviruses are preferred for the in vivo expression of a protein having a lipophilic subtrate transport activity. The adenoviral vectors according to the invention are particularly preferred for direct administration in vivo of a purified suspension and for the ex vivo transformation of cells, in particular autologous cells, in view of their later implantation. Furthermore, the adenoviral vectors according to the invention exhibit, in addition, considerable advantages, such as their very high infection efficiency making it possible to carry out infections using small volumes of viral suspension.

[0488] According to another embodiment of the invention, a line producing retroviral vectors containing the sequence encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins is used for implantation in vivo. The lines that can be used to this end are, for example, the PA317 (U.S. Pat. No. 4,861,719), PsiCrip (WO 90/02806), and GP+envAm-12 (U.S. Pat. No. 5,278,056) cells modified so as to allow the production of a retrovirus containing a nucleic sequence encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins according to the invention. For example, totipotent stem cells, precursors of blood cell lines, may be collected and isolated from a subject. These cells may then be transfected in culture with a retroviral vector containing the sequence encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins under the control of viral promter, a nonviral promter, a promoter specific for macrophages, or under the control of its own promoter. These cells are then reintroduced into the subject. The differentiation of these cells will result in blood cells expressing at least one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins.

[0489] In the vectors of the invention, the sequence encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins may be placed under the control of signals allowing its expression in the infected cells. These may be expression signals that are homologous or heterologous, i.e., signals different from those which are naturally responsible for the expression of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins. They may also be, for example, sequences responsible for the expression of other proteins or synthetic sequences. They may be sequences of eukaryotic or viral genes or derived sequences, which stimulate or repress the transcription of a gene in a specific manner, in a nonspecific manner, or in an inducible manner. By way of example, they may be promoter sequences derived from the genome of the cell which it is desired to infect or from the genome of a virus, for example, the promoters of the E1A or major late promoter (MLP) genes of adenoviruses, the cytomegalovirus (CMV) promoter, the RSV-LTR, and the like. Among the eukaryotic promoters, there may also be mentioned the ubiquitous promoters (HPRT, vimentin, α-actin, tubulin, and the like), the promoters of the intermediate filaments (desmin, neurofilaments, keratin, GFAP, and the like), the promoters of therapeutic genes (MDR, CFTR, factor VIII type, and the like), tissue-specific promoters (pyruvate kinase, villin, promoter of the fatty acid binding intestinal protein, promoter of the smooth muscle cell m-actin, promoters specific for the liver; Apo Al, Apo All, human albumin, and the like) or promoters responding to a stimulus (steroid hormone receptor, retinoic acid receptor, and the like). In addition, these expression sequences may be modified by the addition of enhancer or regulatory sequences and the like. Moreover, when the inserted gene does not contain expression sequences, it may be inserted into the genome of the defective virus downstream of such a sequence.

[0490] In one embodiment, the invention relates to a defective recombinant virus comprising a nucleic acid encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins under the control of a promoter chosen from RSV-LTR or the CMV early promoter.

[0491] As indicated above, the present invention also relates to any use of a virus as described above for the preparation of a composition for the treatment and/or prevention of pathologies linked to the transport of lipophilic substances.

[0492] The present invention also relates to a composition comprising one or more defective recombinant viruses as described above. These compositions may be formulated for administration by the topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, or transdermal route, and the like. Preferably, the compositions of the invention comprise a pharmaceutically-acceptable vehicle or physiologically-compatible excipient for an injectable formulation, for example for an intravenous injection into the subject's portal vein. These may be, for example, isotonic sterile solutions or dry, for example, freeze-dried, compositions that, upon addition of sterilized water or physiological saline as appropriate, allow the preparation of injectable solutions. Direct injection into the subject's portal vein is preferred because it makes it possible to target the infection at the level of the liver and, thus, to concentrate the therapeutic effect at the level of this organ.

[0493] The doses of defective recombinant virus used for the injection may be adjusted as a function of various parameters, for example, as a function of the viral vector, of the mode of administration used, of the relevant pathology or of the desired duration of treatment. In general, the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 10⁴ and 10¹⁴ pfu/ml, and preferably 10⁶ to 10¹⁰ pfu/ml. The term “pfu” (plaque forming unit) corresponds to the infectivity of a virus solution and is determined by infecting an appropriate cell culture and measuring, generally after 48 hours, the number of plaques that result from infected cell lysis. The techniques for determining the pfu titer of a viral solution are well documented in the literature.

[0494] With regard to retroviruses, the compositions according to the invention may directly contain the producing cells with a view to their implantation.

[0495] In this regard, another subject of the invention relates to any mammalian cell infected with one or more defective recombinant viruses according to the invention. For example, the invention encompasses any population of human cells infected with such viruses. These may be cells of blood origin (totipotent stem cells or precursors), fibroblasts, myoblasts, hepatocytes, keratinocytes, smooth muscle and endothelial cells, glial cells, and the like.

[0496] The cells according to the invention may be derived from primary cultures. These may be collected by any technique known to persons skilled in the art and then cultured under conditions allowing their proliferation. With regard to fibroblasts, these may be easily obtained from biopsies, for example, according to the technique described by Ham (1980). These cells may be used directly for infection with the viruses or stored, for example, by freezing, for the establishment of autologous libraries in view of a subsequent use. The cells according to the invention may be secondary cultures obtained, for example, from pre-established libraries (see for example EP 228458, EP 289034, EP 400047, EP 456640).

[0497] The cells in culture are then infected with a recombinant virus according to the invention in order to confer on them the capacity to produce at least one biologically active ABCA5, ABCA6, ABCA9, and ABCA10 protein. The infection is carried out in vitro according to techniques known to persons skilled in the art. For example, depending on the type of cells used and the desired number of copies of virus per cell, persons skilled in the art can adjust the multiplicity of infection and the number of infectious cycles produced. It is clearly understood that these steps must be carried out under appropriate conditions of sterility when the cells are intended for administration in vivo. The doses of recombinant virus used for the infection of the cells may be adjusted by persons skilled in the art according to the desired aim. The conditions described above for administration in vivo may be adapted to infection in vitro. For infection with a retrovirus, it is also possible to co-culture a cell to be infected with a cell producing the recombinant retrovirus according to the invention. This makes it possible to avoid purifying the retrovirus.

[0498] Another subject of the invention relates to an implant comprising mammalian cells infected with one or more defective recombinant viruses according to the invention or cells producing recombinant viruses and an extracellular matrix. Preferably, the implants according to the invention comprise 10⁵ to 10¹⁰ cells. More preferably, they comprise 10⁶ to 10⁸ cells.

[0499] In addition to the extracellular matrix, the implants of the invention may comprise a gelling compound and, optionally, a support allowing the anchorage of the cells.

[0500] For the preparation of the implants according to the invention, various types of gelling agents may be used. The gelling agents are used for the inclusion of the cells in a matrix having the constitution of a gel and, where appropriate, for promoting the anchorage of the cells on the support. Various cell adhesion agents can therefore be used as gelling agents, such as, for example, collagen, gelatin, glycosaminoglycans, fibronectin, lectins, and the like. Preferably, collagen is used in the context of the present invention. This may be collagen of human, bovine, or murine origin. More preferably, type I collagen is used.

[0501] As indicated above, the compositions according to the invention may comprise a support allowing the anchorage of the cells. The term “anchorage” designates any form of biological and/or chemical and/or physical interaction causing the adhesion and/or the attachment of the cells to the support. Moreover, the cells may either cover the support used, penetrate inside this support, or both. It is preferred to use a solid, nontoxic, and/or biocompatible support. For example, it is possible to use polytetrafluoroethylene (PTFE) fibers or a support of biological origin.

[0502] The present invention thus offers a very effective means for the treatment or prevention of pathologies linked to the transport of lipophilic substances.

[0503] In addition, this treatment may be applied to both humans and any animals such as ovines, bovines, domestic animals (dogs, cats and the like), horses, fish, and the like.

[0504] Recombinant Host Cells

[0505] The invention relates to a recombinant host cell comprising a nucleic acid of the invention, for example, a nucleic acid comprising a nucleotide sequence selected from SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence thereof.

[0506] The invention also relates to a recombinant host cell comprising a nucleic acid of the invention, for example, a nucleic acid comprising a nucleotide sequence as depicted in SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence thereof.

[0507] According to another aspect, the invention relates to a recombinant host cell comprising a recombinant vector according to the invention. Therefore, the invention also relates to a recombinant host cell comprising a recombinant vector comprising any of the nucleic acids of the invention, for example, a nucleic acid comprising a nucleotide sequence of selected from SEQ ID NOs: 1-4 and 9-126 or a complementary nucleotide sequence thereof.

[0508] The invention also relates to a recombinant host cell comprising a recombinant vector comprising a nucleic acid comprising a nucleotide sequence as depicted in any one of SEQ ID NOs: 1-4 and 9-126 or of a complementary nucleotide sequence thereof.

[0509] Host cells according to the invention are, for example, the following:

[0510] a) prokaryotic host cells: strains of Escherichia coli (strain DH5-α), of Bacillus subtilis, of Salmonella typhimurium, or strains of genera such as Pseudomonas, Streptomyces and Staphylococus; and

[0511] b) eukaryotic host cells: HeLa cells (ATCC No. CCL2), Cv 1 cells (ATCC No. CCL70), COS cells (ATCC No. CRL 1650), Sf-9 cells (ATCC No. CRL 1711), CHO cells (ATCC No. CCL-61), or 3T3 cells (ATCC No. CRL-6361).

[0512] Methods for Producing ABCA5, ABCA6, ABCA9, and ABCA10 Polypeptides

[0513] The invention also relates to a method for the production of a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-8, said method comprising:

[0514] a) inserting a nucleic acid encoding said polypeptide into an appropriate vector;

[0515] b) culturing, in an appropriate culture medium under conditions allowing the expression of said polypeptide, a previously transformed host cell or transfecting a host cell with the recombinant vector of step a);

[0516] c) recovering the conditioned culture medium or lysing the host cell, for example, by sonication or by osmotic shock;

[0517] d) separating and purifying said polypeptide from said culture medium or, alternatively, from the cell lysates obtained in step c); and

[0518] e) where appropriate, characterizing the recombinant polypeptide produced.

[0519] The polypeptides according to the invention may be characterized by binding to an immunoaffinity chromatography column on which the antibodies directed against this polypeptide or against a fragment or a variant thereof have been previously immobilized.

[0520] According to another aspect, a recombinant polypeptide according to the invention may be purified by passing it over an appropriate series of chromatography columns, according to methods known to persons skilled in the art and described for example in F. Ausubel et al (1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y).

[0521] A polypeptide according to the invention may also be prepared by conventional chemical synthesis techniques either in homogeneous solution or in solid phase. By way of illustration, a polypeptide according to the invention may be prepared by the technique either in homogeneous solution described by Houben Weyl (1974, Methode der Organischen Chemie, E. Wunsch Ed., 15-I:15-II) or the solid phase synthesis technique described by Merrifield (1965, Nature, 207(996):522-523; 1965, Science, 150(693):178-185).

[0522] A polypeptide termed “homologous” to a polypeptide having an amino acid sequence selected from SEQ ID NOs: 5-8 also forms part of the invention. Such a homologous polypeptide comprises an amino acid sequence possessing one or more substitutions of an amino acid by an equivalent amino acid of SEQ ID NOs:5-8.

[0523] An “equivalent amino acid” according to the present invention will be understood to mean, for example, replacement of a residue in the L form by a residue in the D form or the replacement of a glutamic acid (E) by a pyro-glutamic acid according to techniques well known to persons skilled in the art. By way of illustration, the synthesis of a peptide containing at least one residue in the D form is described by Koch (1977). According to another aspect, two amino acids belonging to the same class, that is to say two uncharged polar, nonpolar, basic or acidic amino acids, are also considered as equivalent amino acids.

[0524] Polypeptides comprising at least one nonpeptide bond such as a retro-inverse bond (NHCO), a carba bond (CH₂CH₂), or a ketomethylene bond (CO—CH₂) also form part of the invention.

[0525] The polypeptides according to the invention comprising one or more additions, deletions, substitutions of at least one amino acid generally retain their capacity to be recognized by antibodies directed against the nonmodified polypeptides.

[0526] Antibodies

[0527] The ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides according to the invention, for example, 1) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-8, 2) a polypeptide fragment or variant of a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-8, or 3) a polypeptide termed “homologous” to a polypeptide comprising amino acid sequence selected from SEQ ID NOs: 5-8, may be used for the preparation of an antibody, which may be useful, for example, for detecting the production of a normal or altered form of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides in a patient.

[0528] An antibody directed against a polypeptide termed “homologous” to a polypeptide having an amino acid sequence selected from SEQ ID NOs: 5-8 also forms part of the invention. Such an antibody is directed against a homologous polypeptide comprising an amino acid sequence possessing one or more substitutions of an amino acid by an equivalent amino acid of SEQ ID NOs: 5-8.

[0529] “Antibody” for the purposes of the present invention will be understood to mean in particular polyclonal or monoclonal antibodies or fragments (for example the F(ab)′₂ and Fab fragments) or any polypeptide comprising a domain of the initial antibody recognizing the target polypeptide or polypeptide fragment according to the invention.

[0530] Monoclonal antibodies may be prepared from hybridomas according to the technique described by Kohler and Milstein (1975, Nature, 256:495-497).

[0531] According to the invention, a polypeptide produced recombinantly or by chemical synthesis, and fragments or other derivatives or analogs thereof, including fusion proteins, may be used as an immunogen to generate antibodies that recognize a polypeptide according to the invention. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library. The anti-ABCA5, anti-ABCA6, anti-ABCA9, and anti-ABCA10 antibodies of the invention may be cross reactive, e.g., they may recognize corresponding ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides from different species. Polyclonal antibodies have greater likelihood of cross reactivity. Alternatively, an antibody of the invention may be specific for a single form of any one of ABCA5, ABCA6, ABCA9, and ABCA10. Preferably, such an antibody is specific for any one of human ABCA5, ABCA6, ABCA9, and ABCA10.

[0532] Various procedures known in the art may be used forthe production of polyclonal antibodies to any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides or derivatives or analogs thereof. For the production of antibody, various host animals can be immunized by injection with any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides or a derivatives (e.g., a fragment or fusion protein) thereof, including, but not limited to, rabbits, mice, rats, sheep, goats, etc. In one embodiment, any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides or fragments thereof can be conjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete or incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

[0533] For the preparation of monoclonal antibodies directed toward any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides or fragments, analogs, or derivatives thereof, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include, but are not limited to, the hybridoma technique originally developed by Kohler and Milstein (1975, Nature, 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today, 4:72; Cote et al. 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals (WO 89/12690). In fact, according to the invention, techniques developed for the production of “chimeric antibodies” (Morrison et al., 1984, J. Bacteriol. 159:870; Neuberger et al., 1984, Nature, 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing the genes from a mouse antibody molecule specific for any one of ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides together with genes from a human antibody molecule of appropriate biological activity can be used; such antibodies are within the scope of this invention. Such human or humanized chimeric antibodies are preferred for use in therapy of human diseases or disorders (described infra), since the human or humanized antibodies are much less likely than xenogenic antibodies to induce an immune response, such as an allergic response.

[0534] According to the invention, techniques described for the production of single chain antibodies (U.S. Pat. Nos. 5,476,786 and 5,132,405 to Huston; U.S. Pat. No. 4,946,778) can be adapted to produce ABCA5, ABCA6, ABCA9, and ABCA10 polypeptide-specific single chain antibodies. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al., 1989, Science 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides, its derivatives, or analogs.

[0535] Antibody fragments that contain the idiotype of the antibody molecule can be generated by known techniques. For example, such fragments include, but are not limited to, the F(ab′)₂ fragment, which can be produced by pepsin digestion of the antibody molecule; the Fab′ fragments, which can be generated by reducing the disulfide bridges of the F(ab′)₂ fragment, and the Fab fragments, which can be generated by treating the antibody molecule with papain and a reducing agent.

[0536] In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme, or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labelled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. For example, to select antibodies that recognize a specific epitope of any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides, one may assay hybridomas for a product that binds to any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptide fragments containing such epitope. For selection of an antibody specific to any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides from a particular species of animal, one can select on the basis of positive binding with any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides expressed by or isolated from cells of that species of animal.

[0537] The foregoing antibodies can be used in methods known in the art relating to the localization and activity of any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides, e.g., for western blotting, ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides in situ, measuring levels thereof in appropriate physiological samples, etc. using any of the detection techniques mentioned above or known in the art.

[0538] In a specific embodiment, antibodies that agonize or antagonize the activity of any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides can be generated. Such antibodies can be tested using the assays described infra for identifying ligands.

[0539] The present invention relates to an antibody directed against 1) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-8, 2) a polypeptide fragment or variant of a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-8, or 3) a polypeptide termed “homologous” to a polypeptide comprising amino acid sequence selected from SEQ ID NOS:5-8, also forms part of the invention, as produced in the trioma technique or the hybridoma technique described by Kozbor et al. (1983, Hybridoma, 2(1):7-16).

[0540] The invention also relates to single-chain Fv antibody fragments (ScFv) as described in U.S. Pat. No. 4,946,778 or by Martineau et al. (1998, J Mol Biol, 280(1):117-127).

[0541] The antibodies according to the invention also comprise antibody fragments obtained with the aid of phage libraries as described by Ridder et al., (1995, Biotechnology (NY), 13(3):255-260) or humanized antibodies as described by Reinmann et al. (1997, AIDS Res Hum Retroviruses, 13(11):933-943) and Leger et al., (1997, Hum Antibodies, 8(1):3-16).

[0542] The antibody preparations according to the invention are useful in immunological detection tests intended for the identification of the presence and/or of the quantity of antigens present in a sample.

[0543] An antibody according to the invention may comprise, in addition, a detectable marker that is isotopic or nonisotopic, for example, fluorescent, or may be coupled to a molecule such as biotin according to techniques well known to persons skilled in the art.

[0544] Thus, the subject of the invention is, in addition, a method of detecting the presence of a polypeptide according to the invention in a sample, said method comprising:

[0545] a) bringing the sample to be tested into contact with an antibody directed against 1) a polypeptide. comprising an amino acid sequence of any one of SEQ ID NOs: 5-8, 2) a polypeptide fragment or variant of a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-8, or 3) a polypeptide termed “homologous” to a polypeptide comprising amino acid sequence selected from SEQ ID NOs: 5-8, and

[0546] b) detecting the antigen/antibody complex formed.

[0547] The invention also relates to a box or kit for diagnosis or for detecting the presence of a polypeptide in accordance with the invention in a sample, said box comprising:

[0548] a) an antibody directed against 1) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs:5-8, 2) a polypeptide fragment or variant of a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-8, or 3) a polypeptide termed “homologous” to a polypeptide comprising amino acid sequence selected from SEQ ID NOs: 5-8, and

[0549] b) a reagent allowing the detection of the antigen/antibody complexes formed.

[0550] Compositions and Therapeutic Methods of Treatment

[0551] The invention also relates to compositions intended for the prevention and/or treatment of a deficiency in the transport of cholesterol or inflammatory lipid substances, characterized in that they comprise a therapeutically effective quantity of a polynucleotide capable of giving rise to the production of an effective quantity of at least one of the functional ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides, for example, a polypeptide comprising an amino acid sequence of SEQ ID NOs: 5-8.

[0552] The invention also provides compositions comprising a nucleic acid encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides according to the invention and compositions comprising any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides according to the invention intended for the prevention and/or treatment of diseases linked to a deficiency in the transport of cholesterol or inflammatory lipid substances.

[0553] The present invention also relates to a new therapeutic approach for the treatment of pathologies linked to the transport of lipophilic substances, comprising transferring and expressing in vivo nucleic acids encoding at least one of ABCA5, ABCA6, ABCA9, and ABCA10 proteins according to the invention.

[0554] Thus, the present invention offers a new approach for the treatment and/or the prevention of pathologies linked to abnormalities of the transport of lipophilic substances.

[0555] Consequently, the invention also relates to a composition intended for the prevention of or treatment of subjects affected by a dysfunction in lipophilic substances, comprising a nucleic acid encoding at least one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins in combination with one or more physiologically-compatible vehicles and/or excipients.

[0556] According to a specific embodiment of the invention, a composition is provided for the in vivo production of at least one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins. This composition comprises a nucleic acid encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides placed under the control of appropriate regulatory sequences in solution in a physiologically-acceptable vehicle and/or excipient.

[0557] Therefore, the present invention also relates to a composition comprising a nucleic acid encoding a polypeptide comprising an amino acid sequence of SEQ ID NOs: 5-8, wherein the nucleic acid is placed under the control of appropriate regulatory elements. Such a composition may comprise a nucleic acid comprising a nucleotide sequence of SEQ ID NOs:1-4, placed under the control of appropriate regulatory elements.

[0558] According to another aspect, the subject of the invention is also a preventive and/or curative therapeutic method of treating diseases caused by a deficiency in the transport of lipophilic substances, such a method comprising administering to a patient a nucleic acid encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides according to the invention in said patient, said nucleic acid being, where appropriate, combined with one or more physiologically-compatible vehicles and/or excipients.

[0559] The invention also relates to a composition intended for the prevention of or treatment of subjects affected by a dysfunction in the transport of lipophilic substances, comprising a recombinant vector according to the invention in combination with one or more physiologically-compatible excipients.

[0560] According to one embodiment, a method of introducing a nucleic acid according to the invention into a host cell, for example, a host cell obtained from a mammal, in vivo, comprises a step during which a preparation comprising a pharmaceutically-compatible vector and a “naked” nucleic acid according to the invention, placed under the control of appropriate regulatory sequences, is introduced by local injection at the site of the chosen tissue, for example, a smooth muscle tissue, the “naked” nucleic acid being absorbed by the cells of this tissue.

[0561] The invention also relates to the use of a nucleic acid according to the invention, encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins, for the manufacture of a medicament intended for the prevention and/or treatment in various forms or more particularly for the treatment of subjects affected by a dysfunction in the transport of lipophilic substances.

[0562] The invention also relates to the use of a recombinant vector according to the invention, comprising a nucleic acid encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins, for the manufacture of a medicament intended for the prevention and/or treatment of subjects affected by a dysfunction in the transport of lipophilic substances.

[0563] As indicated above, the present invention also relates to the use of a defective recombinant virus according to the invention for the preparation of a composition for the treatment and/or prevention of pathologies linked to the transport of lipophilic substances.

[0564] The invention relates to the use of such a defective recombinant virus for the preparation of a composition intended for the treatment and/or prevention of a deficiency associated with the transport of lipophilic substances. Thus, the present invention also relates to a composition comprising one or more defective recombinant viruses according to the invention.

[0565] The present invention also relates to the use of cells genetically modified ex vivo with a virus according to the invention and to producing cells such viruses, implanted in the body, allowing a prolonged and effective expression in vivo of at least one biologically active ABCA5, ABCA6, ABCA9, and ABCA10 protein.

[0566] The present invention shows that it is possible to incorporate a nucleic acid encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides into a viral vector, and that these vectors make it possible to effectively express a biologically active, mature form of the encoded protein. More particularly, the invention shows that the in vivo expression of any one of the ABCA5, ABCA6, ABCA9, and ABCA10 genes may be obtained by direct administration of an adenovirus or by implantation of a producing cell or of a cell genetically modified by an adenovirus or by a retrovirus incorporating such a DNA.

[0567] The compositions of the invention may comprise a pharmaceutically-acceptable vehicle or physiologically-compatible excipient for an injectable formulation, for example, for an intravenous injection into the subject's portal vein. These may be, for example, isotonic sterile solutions or dry, for example, freeze-dried, compositions which, upon addition of sterilized water or physiological saline, as appropriate, allow the preparation of injectable solutions. Direct injection into the subject's portal vein is preferred because it makes it possible to target the infection at the level of the liver and, thus, to concentrate the therapeutic effect at the level of this organ.

[0568] The term “pharmaceutically-acceptable vehicle or excipient” includes diluents and fillers that are pharmaceutically-acceptable for method of administration, are sterile, and may be aqueous or oleaginous suspensions formulated using suitable dispersing or wetting agents and suspending agents. The particular pharmaceutically-acceptable carrier and the ratio of active compound to carrier are determined by the solubility and chemical properties of the composition, the particular mode of administration, and standard pharmaceutical practice.

[0569] Any nucleic acid, polypeptide, vector, or host cell of the invention may be introduced in vivo in a pharmaceutically-acceptable vehicle or excipient. The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that are physiologically-tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and, more particularly, in humans. The term “excipient” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions may be employed as excipients, particularly for injectable solutions. Suitable pharmaceutical excipients are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.

[0570] The pharmaceutical compositions according to the invention may be equally well administered by the oral, rectal, parenteral, intravenous, subcutaneous, or intradermal route.

[0571] According to another aspect, the subject of the invention is also a preventive and/or curative therapeutic method of treating diseases caused by a deficiency in the transport of cholesterol or inflammatory lipid substances, comprising administering to a patient or subject a nucleic acid encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides, said nucleic acid being combined with one or more physiologically-compatible vehicles and/or excipients.

[0572] In another embodiment, the nucleic acids, recombinant vectors, and compositions according to the invention can be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science, 249:1527-1533; Treat et al., 1989, Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss: New York, pp. 353-365; and Lopez-Berestein, 1989, In: Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss: New York, pp. 317-327).

[0573] In a further aspect, recombinant cells that have been transformed with a nucleic acid according to the invention and that express high levels of any one of ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides according to the invention can be transplanted in a subject in need of any one of ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides. Preferably autologous cells transformed with an any one of ABCA5, ABCA6, ABCA9, and ABCA10 encoding nucleic acids according to the invention are transplanted to avoid rejection; alternatively, technology is available to shield non-autologous cells that produce soluble factors within a polymer matrix that prevents immune recognition and rejection.

[0574] A subject in whom administration of the nucleic acids, polypeptides, recombinant vectors, recombinant host cells, and compositions according to the invention is performed is preferably a human, but can be any animal. Thus, as can be readily appreciated by one of ordinary skill in the art, the methods and pharmaceutical compositions of the present invention are particularly suited to administration to any animal, particularly a mammal, and including, but by no means limited to, domestic animals, such as feline or canine subjects, farm animals, such as but not limited to bovine, equine, caprine, ovine, and porcine subjects, wild animals (whether in the wild or in a zoological garden), research animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., avian species, such as chickens, turkeys, songbirds, etc., i.e., for veterinary medical use.

[0575] Preferably, a pharmaceutical composition comprising a nucleic acid, a recombinant vector, or a recombinant host cell, as defined above, will be administered to the patient or subject.

[0576] Mehtods of Screening an Agonist or Antagonist Compound for the ABCA5, ABCA6, ABCA9, and ABCA10 Polypeptides

[0577] According to another aspect, the invention also relates to various methods of screening compounds or small molecules for therapeutic use which are useful in the treatment of diseases due to a deficiency in the transport of cholesterol or inflammatory lipid substances.

[0578] The invention therefore also relates to the use of any one of ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides, or of cells expressing any one of ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides, for screening active ingredients for the prevention and/or treatment of diseases resulting from a dysfunction in ABCA5, ABCA6, ABCA9, or ABCA10 defiencies. The catalytic sites and oligopeptide or immunogenic fragments of any one of ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides can serve for screening product libraries by a whole range of existing techniques. The polypeptide fragment used in this type of screening may be free in solution, bound to a solid support, at the cell surface or in the cell. The formation of the binding complexes between any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptide fragments and the tested agent can then be measured.

[0579] Another product screening technique which may be used in high-flux screenings giving access to products having affinity for the protein of interest is described in application WO84/03564. In this method, applied to any one of ABCA5, ABCA6, ABCA9, and ABCA10 proteins, various products are synthesized on a solid surface. These products react with corresponding ABCA5, ABCA6, ABCA9, and ABCA10 proteins or fragments thereof and the complex is washed. The products binding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins are then detected by methods known to persons skilled in the art. Non-neutralizing antibodies can also be used to capture a peptide and immobilize it on a support.

[0580] Another possibility is to perform a product screening method using any one of the ABCA5, ABCA6, ABCA9, and ABCA10 neutralizing competition antibodies, at least one of ABCA5, ABCA6, ABCA9, and ABCA10 proteins and a product potentially binding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins. In this manner, the antibodies may be used to detect the presence of a peptide having a common antigenic unit with any one of ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides or proteins.

[0581] Of the products to be evaluated for their ability to increase activity of any one of ABCA5, ABCA6, ABCA9, and ABCA10, there may be mentioned in particular kinase-specific ATP homologs involved in the activation of the molecules, as well as phosphatases, which may be able to avoid the dephosphorylation resulting from said kinases. There may be mentioned in particular inhibitors of the phosphodiesterase (PDE) theophylline and 3-isobutyl-1-methylxanthine type or the adenylcyclase forskolin activators.

[0582] Accordingly, this invention relates to the use of any method of screening products, i.e., compounds, small molecules, and the like, based on the method of translocation of cholesterol or lipophilic substances between the membranes or vesicles, this being in all synthetic or cellular types, that is to say of mammals, insects, bacteria, or yeasts expressing constitutively or having incorporated any one of human ABCA5, ABCA6, ABCA9, and ABCA10 encoding nucleic acids. To this effect, labeled lipophilic substances analogs may be used.

[0583] Furthermore, knowing that the disruption of numerous transporters have been described (van den Hazel et al., 1999, J. Biol Chem, 274: 1934-41), it is possible to think of using cellular mutants having a characteristic phenotype and to complement the function thereof with at least one of ABCA5, ABCA6, ABCA9, and ABCA10 proteins and to use the whole for screening purposes.

[0584] The invention also relates to a method of screening a compound or small molecule active on the transport of lipophilic substances, an agonist or antagonist of any one of ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides, said method comprising the following steps:

[0585] a) preparing a membrane vesicle comprising at least one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides and a lipid substrate comprising a detectable marker;

[0586] b) incubating the vesicle obtained in step a) with an agonist or antagonist candidate compound;

[0587] c) qualitatively and/or quantitatively measuring release of the lipid substrate comprising a detectable marker; and

[0588] d) comparing the release measurement obtained in step b) with a measurement of release of labeled lipophilic substrate by a vesicle that has not been previously incubated with the agonist or antagonist candidate compound.

[0589] ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides comprise an amino acid sequence selected from SEQ ID NOs: 5-8.

[0590] According to a first aspect of the above screening method, the membrane vesicle is a synthetic lipid vesicle, which may be prepared according to techniques well known to a person skilled in the art. According to this particular aspect, ABCA5, ABCA6, ABCA9, and ABCA10 proteins may be recombinant proteins.

[0591] According to a second aspect, the membrane vesicle is a vesicle of a plasma membrane derived from cells expressing at least one of ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides. These may be cells naturally expressing any one of ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides or cells transfected with a nucleic acid encoding at least one ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides or recombinant vector comprising a nucleic acid encoding any one of ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides.

[0592] According to a third aspect of the above screening method, the lipid substrate is chosen from prostaglandins or prostacyclins.

[0593] According to a fourth aspect of the above screening method, the lipid substrate is chosen from cholesterol or phosphatidylcholine.

[0594] According to a fifth aspect, the lipid substrate is radioactively labelled, for example with an isotope chosen from ³H or ¹²⁵I.

[0595] According to a sixth aspect, the lipid substrate is labelled with a fluorescent compound, such as NBD or pyrene.

[0596] According to a seventh aspect, the membrane vesicle comprising the labelled lipophilic substances and any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides is immobilized at the surface of a solid support prior to step b).

[0597] According to a eighth aspect, the measurement of the fluorescence or of the radioactivity released by the vesicle is the direct reflection of the activity of lipid substrate transport by any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides.

[0598] The invention also relates to a method of screening a compound or small molecule active on the transport of cholesterol or lipid substances, an agonist or antagonist of any one of ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides, said method comprising the following steps:

[0599] a) obtaining cells, for example a cell line, that, either naturally or after transfecting the cell with any one of ABCA5, ABCA6, ABCA9, AND ABCA10 encoding nucleic acids, expresses any one of ABCA5, ABCA6, ABCA9, AND ABCA10 polypeptides;

[0600] b) incubating the cells of step a) in the presence of an anion labelled with a detectable marker;

[0601] c) washing the cells of step b) in order to remove the excess of the labelled anion which has not penetrated into these cells;

[0602] d) incubating the cells obtained in step c) with an agonist or antagonist candidate compound for any one of ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides;

[0603] e) measuring efflux of the labelled anion; and

[0604] f) comparing the value of efflux of the labelled anion determined in step e) with a value of the efflux of a labelled anion measured with cells that have not been previously incubated in the presence of the agonist or antagonist candidate compound of any one of ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides.

[0605] In a first specific embodiment, any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides comprise an amino acid sequence of SEQ ID NOs: 5-8.

[0606] According to a second aspect, the cells used in the screening method described above may be cells not naturally expressing, or alternatively expressing at a low level, any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides, said cells being transfected with a recombinant vector according to the invention capable of directing the expression of a nucleic acid encoding any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides.

[0607] According to a third aspect, the cells may be cells having a natural deficiency in anion transport, or cells pretreated with one or more anion channel inhibitors such as Verapamil™ or tetraethylammonium.

[0608] According to a fourth aspect of said screening method, the anion is a radioactively labelled iodide, such as the salts K¹²⁵I or Na¹²⁵I.

[0609] According to a fifth aspect, the measurement of efflux of the labelled anion is determined periodically over time during the experiment, thus making it possible to also establish a kinetic measurement of this efflux.

[0610] According to a sixth aspect, the value of efflux of the labelled anion is determined by measuring the quantity of labelled anion present at a given time in the cell culture supernatant.

[0611] According to a seventh aspect, the value of efflux of the labelled anion is determined as the proportion of radioactivity found in the cell culture supernatant relative to the total radioactivity corresponding to the sum of the radioactivity found in the cell lysate and the radioactivity found in the cell culture supernatant.

[0612] The subject of the invention is also a method of screening a compound or small molecule active on the metabolism of lipophilic substances, an agonist or antagonist of any one of ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides, said method comprising the following steps:

[0613] a) culturing cells of a human myocyte line in an appropriate culture medium, in the presence of purified human albumin;

[0614] b) incubating the cells of step a) simultaneously in the presence of a compound stimulating the production of interleukine and of an agonist or antagonist candidate compound;

[0615] c) incubating the cells obtained in step b) in the presence of an appropriate concentration of ATP;

[0616] d) measuring interleukinereleased into the cell culture supernatant; and

[0617] e) comparing the value of the release of the interleukineobtained in step d) with the value of the interleukinereleased into the culture supernatant of cells which have not been previously incubated in the presence of the agonist or antagonist candidate compound.

[0618] According to a first aspect of the screening method described above, the cells used belong to the human or mousemyocytes.

[0619] According to a second aspect of the screening method, the compound stimulating the production of interleukineis a lipopolysaccharide.

[0620] According to a third aspect of said method, the production of all interleukinesand TNF alpha by these cells is also qualitatively and/or quantitatively determined.

[0621] According to a fourth aspect, the level of expression of the messenger RNA encoding interleukineis also determined.

[0622] The following examples are intended to further illustrate the present invention but do not limit the invention.

EXAMPLES Example 1 Search of Human ABCA5, ABCA6, ABCA9, and ABCA10 Genes in Genomic Database

[0623] Expressed sequence tags (EST) of ABCA1-like genes as described by Allikmets et al. (Hum Mol Genet, 1996, 5, 1649-1655) were used to search Genbank and UniGene nucleotide sequence databases using BLAST2 (Altschul et al, 1997, Nucleic Acids Res., 25:3389-3402). The main protein sequences databases screened were Swissprot, TrEMBL, Genpept, and PIR.

[0624] The genomic DNA analysis was performed by combination of several gene-finding programs such as GENSCAN (Burge and Karlin, 1997, J Mol Biol.; 268(1):78-94), FGENEH/FEXH (Solovyev and Salamov, 1997, lsmb; 5:294-302), and XPOUND (Thomas and Skolnick, 1994, J Math Appl Med Biol.;1 1(1):1-16). The combination of different tools lead to increase sensitivity and specificity. The second step in the genomic DNA analysis is the homology searching in the EST and protein databases. Combination of software performing database searching and software for exon/intron prediction give the best sensitive and specific results. Sequence assembly and analysis were performed using the Genetics Computer Group (GCG) sequence analysis software package.

[0625] Multiple alignments were generated by GAP software from GCG package and the Dialign2 program (Morgenstern et al, 1996, Proc Natl Acad Sci U S A.; 93(22):12098-103), the FASTA3 package (Pearson and Lipman, 1988, Proc Natl Acad Sci U S A.; 85(8):2444-8) and SIM4 (Florea et al, 1998, Genome Res. 1998 Sep. 8, 1998;(9):967-74). The specific ABCA motifs used in our process were the TMN, TMC, NBDI and NBD2 described in the literature (Broccardo et al, 1999). This corresponds in ABCA1 to residues 630-846 for the N terminal (TMN=exon 14-16) and from 1647-1877 for the C terminal set of membrane spanners (TMC=exon 36-40). The NBD corresponds to the extended nucleotide binding domain, i.e., in ABCA1 it spans from amino acids 885-1152 for the N-terminal one (NBDI=exon 18-22) and 1918-2132 for the C-terminal one (NBD2 =exon 42-47).

[0626] Sequence comparison between candidate ABCA ESTs with two overlapping BAC clones containing the microsatellite marker D17S940 (GenBank accession #AC005495, AC005922, revealed surprisingly that all these ESTs are located within this 325,000 bp. An electronic intron/exon prediction was performed by using the AC005495 and AC005922 BAC sequences, and provided transcript sequences which were predictied to correspond to the full coding sequence (CDS) of ABCA6 and ABCA9. ABCA5 gene sequences were found to be partially contain in the contig of BACs as 3′ and 5′ ends, respectively. Moreover, the analysis of the sequence revealed the ABCA10 gene.

[0627] Additional sequence information for ABCA5 was obtained by using the working draft BACs that overlap with the above described BAC contig (AC005495 and AC005922) on both 3′ and 5′ ends. A supplemental BAC working draft, I., GenBank Accession number AC007763, was then identified on the 5′ end. A parallel database mining approach based on the specific motifs search in the different Genbank subdivisions and UniGene Homo sapiens led to identification of two of these sequences (one contains a TMC motif, one contains a NBDL domain) which matched with two fragments of the BAC #AC007763.

[0628] Using exon-intron sequence of these genes, we compared the sequence of the cDNAs with the genomic sequence of the BACs (AC005495, AC005922 and AC007763) and established the approximate genomic size and respective orientation of the ABCA5, ABCA6, ABCA9, and ABCA10 genes.

Example 2 5′ Extension of the Human ABCA5, ABCA6, ABCA9, and ABCA10 cDNA

[0629] This Example describes the isolation and identification of cDNA molecules encoding the full length human ABCA5, ABCA6, ABCA9, and ABCA10 protein. 5′ extension of the partial ABCA5, ABCA6, ABCA9, and ABCA10 cDNA sequence was performed by using a combination of 5′ RACE and RT-PCR on liver, heart, or testis.

[0630] Oligonucleotide primers allowing to distinguish novel ABCA5-6 and 9-10 genes from other family members, were designed taking advantage of the exonic/intronic prediction of the genomic sequence and used to identify specific cDNA transcript by RT-PCR on RNA from various human tissues. With the exception of ABCA10 that required an additional cloning step, all RT-PCR products were directly sequenced. In the case of ABCA6, ABCA9, and ABCA10 a 5′ RACE step was also performed in order to confirm the initiator ATG codon. The identification of the full CDS of ABCA5 was obtained by linking the 3 potential fragments of the transcript by RT-PCR and direct sequencing. Finally, full ORF sequences of these new genes that belong to the same chromosome 17 cluster were determined.

[0631] Reverse Transcription

[0632] In a total volume of 11.5 μl, 500 ng of mRNA poly(A)+(Clontech) mixed with 500 ng of oligodt are denaturated at 70° C. for 10 min and then chilled on ice. After addition of 10 units of RNAsin, 10 mM DTT, 0.5 mM dNTP, Superscript first strand buffer and 200 units of Superscript II (Life Technologies), the reaction is incubated for 45 min at 42° C. We used poly(A) mRNA from liver, heart, brain and lung for ABCA9, from testis for ABCA5, from testis and heart for ABCA10.

[0633] PCR

[0634] Each polymerase chain reaction contained 400 μM each dNTP, 2 units of Thermus aquaticus (Taq) DNA polymerase (Ampli Taq Gold; Perkin Elmer), 0.5 μM each primer, 2.5 mM MgCl₂, PCR buffer and 50 ng of DNA, or about 25 ng of cDNA, or 1/50e of primary PCR mixture. Reactions were carried out for 30 cycles in a Perkin Elmer 9700 thermal cycler in 96-well microtiter plates. After an initial denaturation at 94° C. for 10 min, each cycle consisted of: a denaturation step of 30 s (94° C.), a hybridization step of 30 s (64° C. for 2 cycles, 61° C. for 2 cycles, 58° C. for cycles and 55° C. for 28 cycles), and an elongation step of 1 min/kb (72° C.). PCR ended with a final 72° C. extension of 7 min. In case of RT-PCR, control reactions without reverse transcriptase and reactions containing water instead of cDNA were performed for every sample.

[0635] DNA Sequencing

[0636] PCR products are analyzed and quantified by agarose gel electrophoresis, purified with a P100 column. Purified PCR products were sequenced using ABI Prism BigDye terminator cycle sequencing kit (Perkin Elmer Applied Biosystems). The sequence reaction mixture was purified using Microcon-100 microconcentrators (Amicon, Inc., Beverly). Sequencing reactions were resolved on an ABI 377 DNA sequencer (Perkin Elmer Applied Biosystems) according to manufacturer's protocol (Applied Biosystems, Perkin Elmer).

[0637] 5′ Rapid Amplification of cDNA Ends (RACE)

[0638] 5′ RACE analysis was performed using the SMART RACE cDNA amplification kit (Clontech, Palo Alto, Calif.). Human testis, liver and heart polyA+RNA (Clontech) were used as template to generate the 5′ SMART cDNA library according to the manufacturer's instructions. First-amplification primers and nested primers were designed from the cDNA sequence. Amplimers of the nested PCR were cloned. Insert of specific clones are amplified by PCR with universal primers (Rev and −21) and sequenced on both strands. Primers ABC-A6_L1, L2, ABC-A9_L1, L2, and ABC-A10_L1, L2 were used to identify 5′ ends of ABC-A6, ABC-A9, and ABC-A10 respectively.

[0639] Primers

[0640] Oligonucleotides were selected using Prime from GCG package or Oligo 4 (National Biosciences, Inc.) softwares. Primers were ordered from Life Technologies, Ltd and used without further purification (Table 15). TABLE 15 RT-PCR and 5′RACE primers SEQ ID NO: Name Sequence Orientation 127 ABCA5_A CAGTGACTATGTATCCGTG Forward 128 ABCA5_B GATGGTTTCTCCTCACAAC Reverse 129 ABCA5_C CACCAGACAATGAGGATGA Forward 130 ABCA5_D GCTATATTCTTCAATGGCA Reverse 131 ABCA5_E CCTAGAAGTAGACCGCCTT Forward 132 ABCA5_G GTTGTGAGGAGAAACCATC Forward 133 ABCA5_H CTGGATGGTTTCAGTCACA Reverse 134 345770_A CAGAAAAGCCAATCGGGTG Forward 135 345770_B CCAGGTATATGTTGTTTAACCAG Reverse 136 345770_C GGGTCAGATTACTGCCTTAC Forward 137 345770_D GAACATTGAAGAACCAACAC Reverse 138 345770_F GTAAGGCAGTAATCTGACCC Reverse 139 445188_B GGAAACTGGACAGAATGC Reverse 140 445188_C CTACCCTATTTCACATGCC Forward 141 445188_D GTTTCTCCCATAATAACAGC Reverse 142 445188_E GCTGTTATTATGGGAGAAAC Forward 143 445188_L1 AGACTACAGTAACAAAAGCCTAGTGCAGCC Reverse 144 445188_L2 ATCCAATCCTATTAGTGTGACAAAGGCTTG Reverse 145 ABCA6_A TCAGCAAACCAAAGCACTTC Forward 146 ABCA6_B TGACATCAACTCCTCCATCAC Reverse 149 ABCA6_C CCCTGTGATGGAGGAGTTG Forward 148 ABCA6_C1 TCATTGCTGGGATGGATATG Forward 154 ABCA6_D AAGGGTCAGGAAAAATTACACC Reverse 151 ABCA6_D1 GAATGCTGAATCTTGGAGAC Reverse 153 ABCA6_E TGGTGTAATTTTTCCTGACCC Forward 152 ABCA6_E1 GATTCAGATTATCAAACTGG Forward 157 ABCA6_F CCACTTCCTTTAGATGAATCCC Reverse 155 ABCA6_F1 GGAATTCAGGAGCTACTGG Reverse 158 ABCA6_G AAGTGGAACAAGAGGTACAACG Forward 156 ABCA6_G1 GATTGTCTGTTCCAACAGAAGG Forward 160 ABCA6_H GGGGATGTGATGAGTAATGAAG Reverse 159 ABCA6_H1 ATGGTAATCCCAAAAGTCAGC Reverse 161 ABCA6_I CTTCATTACTCATCACATCCCC Forward 163 ABCA6_J GATCAACAGGCTGGTACGG Reverse 162 ABCA6_K ACAACTTCCCCAGGAACCC Forward 165 ABCA6_L TGCCCACACCAGTAAGCAG Reverse 164 ABCA6_M CAAGAAAAATGCTAAGTCCCAG Forward 166 ABCA6_N GAAAATCAGTGGCACTCAATTC Reverse 167 ABCA6_O TGCCACTGATTTTCTAGTCTGC Forward 169 ABCA6_P CCTTTCAGTTCCACCTCTCC Reverse 168 ABCA6_Q CTGGGATCACAAAGCCAAC Forward 171 ABCA6_R AATACCTTTCCTGCCCTGC Reverse 170 ABCA6_S TCCACACTGAGATTCTGAAGC Forward 172 ABCA6_T GCCTGACTCTTTGGGTGAC Reverse 147 ABCA6_L1 GTACATGAAAACTCACCATATCCATCCC Reverse 146 ABCA6_L2 GCAAGTGCTGTTTTATTCATTATCTGCTG Reverse 173 ABCA9_A TGAGCGTGGGTCAGCAAAC Forward 174 ABCA9_B GCAACTCCTCCTTGGGCAAC Reverse 175 ABCA9_C TTTGTTGCCCAAGGAGGAG Forward 176 ABCA9_D GGAAAAACAAGGGAGAACATCG Reverse 177 ABCA9_E1 GCCCACTTGGATTCTTCAC Forward 178 ABCA9_F1 CCACACCTTTCAAAGCTTCTAC Reverse 179 ABCA9_G1 ATGTGGTCCTTGAGAATGAAAC Forward 180 ABCA9_G2 ACTGTGAAAGAAAACCTCAGGC Forward 181 ABCA9_H1 CTTCATGTGGCAAAATCCC Reverse 182 ABCA9_H2 TGTGCTGTCAATTTGGCATC Reverse 183 ABCA9_I AAGAAGAAATGGGGCATAGG Forward 184 ABCA9_J TGTATTTGGAGACAGTTCCCAC Reverse 185 ABCA9_K1 AACAATCAGTGGCGTGGCG Forward 202 ABCA9_L1(RACE) CTTGGGTAGTTTTGGATTCAGGTGC Reverse 186 ABCA9_L1(CV) GACATCCAGGAGGACAGGAAAG Reverse 203 ABCA9_L2 AGATCCATTGAAGACATTTGAGGAGTG Reverse 187 ABCA9_M GCAGCCTCTTTCACTCCATAC Forward 188 ABCA9_M1 CATTGTGTCAGGTGATGAAAAG Forward 189 ABCA9_N TTCATTTCTAGGCATCGCAG Reverse 190 ABCA9_N1 CATTAGCAGGAGGATCAAAAAG Reverse 191 ABCA9_O TCTAGGGCTATTTTTTGGCAC Forward 192 ABCA9_P CGCTCCCTTTCAAAATCAC Reverse 193 ABCA9_Q1 TGCGAGACTTTGATGAGACAC Forward 194 ABCA9_T2 AGACCATCAGGGAGGAGAAC Reverse 195 ABCA9_U TGTGCCAGCAACCAAATC Forward 196 ABCA9_U1 GCTGGAGATGAAGCTGAAGAAC Forward 201 ABCA9_U2 AAGCATGATGTAGTAGTGACCC Forward 197 ABCA9_V1 TTTCCACTTCACCGAGGG Reverse 198 ABCA9_W CCATGTTTTGTCTGTTGTGCC Forward 199 ABCA9_X CACCCATCAACCCATCATCTAC Reverse 200 ABCA9_Z AGGCACAACAGACAAAACATGG Reverse 204 ABCA10_A GATTGACATACATTTGCTTC Forward 205 ABCA10_B TACAGTGAAGAGAAATCCAG Reverse 206 ABCA10_C TGGAATTAGACATGCAAA Forward 207 ABCA10_D TGAAGAGGATAAGTCGGTC Reverse 208 ABCA10_E TATAATCGCTGATGCTGC Reverse 212 ABCA10_I AGATAAGCGTGCGTCAAC Forward 215 ABCA10_N TCATCAACATTTCCCAGC Reverse 216 ABCA10_AA GAAATACTGGAGATGAGTCTG Forward 217 ABCA10_AB GAGCTTAAGAGCTTCCACC Reverse 213 ABCA11_C TCTTATGGGAATTGTTAGCA Forward 214 ABCA11_H TTATGACTGGTTCCTCCTC Reverse 209 ABCA10_L1 ACCAGGCCAGAGTCATTAAACTGATC Reverse 210 ABCA10_L2 CCGAAAAGATGCACAAATATAGCCC Reverse 211 ABCA10_U2 CTCAAAACTTCATTCTAATTGTGCCC Forward

Example 3 Tissue Distribution of the Transcripts of the ABCA5, ABCA6, ABCA9, and ABCA10 Genes According to the invention.

[0641] The profile of expression of the polynucleotides according to the present invention is determined according to the protocols for PCR-coupled reverse transcription and Northern blot analysis described in particular by Sambrook et al. (1989, Molecular cloning: a laboratory manual. 2ed. Cold Spring Harbor Laboratory, Cold spring Harbor, N.Y.).

[0642] For example, in the case of an analysis by reverse transcription, a pair of primers as described above may be synthesized from a cDNA of the human ABCA5, ABCA6, ABCA9, and ABCA10 genes. This primer pair may be used to detect the corresponding ABCA5, ABCA6, ABCA9, and ABCA10 cDNAs.

[0643] The polymerase chain reaction (PCR) is carried out on cDNA templates corresponding to retrotranscribed polyA⁺mRNAs (Clontech). The reverse transcription to cDNA is carried out with the enzyme SUPERSCRIPT II (GibcoBRL, Life Technologies) according to the conditions described by the manufacturer. The polymerase chain reaction is carried out according to standard conditions, in 20 μl of reaction mixture with 25 ng of cDNA preparation. The reaction mixture is composed of 400 μM of each of the dNTPs, 2 units of Thermus aquaticus (Taq) DNA polymerase (Ampli Taq Gold; Perkin Elmer), 0.5 μM of each primer, 2.5 mM MgCl2, and PCR buffer. Thirty four PCR cycles [denaturing 30 seconds at 94° C., annealing of 30 seconds divided up as follows during the 34 cycles: 64° C. (2 cycles), 61° C. (2 cycles), 58° C. (2 cycles), and 55° C. (28 cycles), and an extension of one minute per kilobase at 72° C.] are carried out after a first step of denaturing at 94° C. for 10 minutes using a Perkin Elmer 9700 thermocycler. The PCR reactions are visualized on agarose gel by electrophoresis. The cDNA fragments obtained may be used as probes for a Northern blot analysis and may also be used for the exact determination of the nucleotide sequence.

[0644] Northern Blot Analysis

[0645] To study mRNA expression of the ABCA5, ABCA6, ABCA9, and ABCA10 genes, human MTN (Multiple Tissue Northern) blots (Human II 7759-1, Human 7760-1, and human fetal II 7756-1, Clontech) were hybridized with specific pools of probes consisting in two amplimers: ABCA5_A-ABCA5_B and 445188_C-445188_D for ABC-A5, ABCA6_A-ABCA6_B and ABCA6_S-ABCA6_T for ABC-A6, ABCA9_A-ABCA9-B and ABCA9_M1-ABCA9_N1 for ABC-A9. A unique RT-PCR product obtained between ABCA10_I-ABCA10_B was used for ABC-A10 (Table 15).

[0646] Preparation of the Probe

[0647] PCR products were gel-purified using Qiaquick® column (Qiagen). 10-20 ng of purified PCR product were radiolabelled with [α³²P]dCTP (Amersham; 6000 Ci/mmol, 10 mCi/ml) by the random priming method (Rediprime kit; Amersham) according to the manufacturer's protocol. Unincorporated radioactive nucleotides were separated from the labelled probe by filtration on a G50 microcolumn (Pharmacia). Probe was competed with 50 μg of denatured human Cot1 DNA during 2 hours at 65° C.

[0648] Hybridization

[0649] Prehybridization of Northern blot (6 hours at 42° C.) with hybridization solution (5×SSPE, 5×Denhardt's, 2.5% Dextran, 0.5% SDS, 50% formamide, 100 μg/ml denaturated salmon sperm DNA, 40 μg denatured human DNA) was followed by hybridization with radiolabelled probe (2.10⁶ cpm/ml hybridization solution) and 40 pg of denatured human DNA. Filters were washed in 2×SSC for 30 min at room temperature, twice in 2×SSC-0.1% SDS for 10 min at 65° C. and twice in 1×SSC-0.1% SDS for 10 min at 65° C. Northern blot were analyzed after overnight exposure on the Storm (Molecular Dynamics, Sunnyvale, Calif.). The human transferrin probe was used to control the amount of RNA in each lane of the membrane.

Example 4 Construction of the Expression Vector Containing the Complete cDNA of ABCA5, ABCA6, ABCA9, or ABCA10 in Mammalian Cells

[0650] The ABCA5, ABCA6, ABCA9, or ABCA10 genes may be expressed in mammalian cells. A typical eukaryotic expression vector contains a promoter which allows the initiation of the transcription of the mRNA, a sequence encoding the protein, and the signals required for the termination of the transcription and for the polyadenylation of the transcript. It also contains additional signals such as enhancers, the Kozak sequence and sequences necessary for the splicing of the mRNA. An effective transcription is obtained with the early and late elements of the SV40 virus promoters, the retroviral LTRs or the CMV virus early promoter. However, cellular elements such as the actin promoter may also be used. Many expression vectors may be used to carry out the present invention, an example of such a vector is pcDNA3 (Invitrogen).

Example 5 Production of Normal and Mutated ABCA5, ABCA6, ABCA9, or ABCA10 Polypeptides

[0651] The normal ABCA5, ABCA6, ABCA9, or ABCA10 polypeptides encoded by complete corresponding cDNAs whose isolation is described in Example 2, or the mutated ABCA5, ABCA6, ABCA9, or ABCA10 polypeptides whose complete cDNA may also be obtained according to the techniques described in Example 2, may be easily produced in a bacterial or insect cell expression system using the baculovirus vectors or in mammalian cells with or without the vaccinia virus vectors. All the methods are now widely described and are known to persons skilled in the art. A detailed description thereof will be found for example in F. Ausubel et al. (1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y).

Example 6 Production of an Antibody Directed Against One of the Mutated ABCA5, ABCA6, ABCA9, or ABCA10 Polypeptides

[0652] The antibodies in the present invention may be prepared by various methods (Current Protocols In Molecular Biology Volume 1 edited by Frederick M. Ausubel, Roger Brent, Robert E. Kingston, David D. Moore, J. G. Seidman, John A. Smith, Kevin Struhl—Massachusetts General Hospital Harvard Medical School, chapter 11, 1989). For example, the cells expressing a polypeptide of the present invention are injected into an animal in order to induce the production of serum containing the antibodies. In one of the methods described, the proteins are prepared and purified so as to avoid contaminations. Such a preparation is then introduced into the animal with the aim of producing polyclonal antisera having a higher activity.

[0653] In the preferred method, the antibodies of the present invention are monoclonal antibodies. Such monoclonal antibodies may be prepared using the hybridoma technique (Köhler et al, 1975, Nature, 256:495; Köhler et al, 1976, Eur. J. Immunol. 6:292; Köhler et al, 1976, Eur. J. Immunol., 6:511; Hammeling et al., 1981, Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681). In general, such methods involve immunizing the animal (preferably a mouse) with a polypeptide or better still with a cell expressing the polypeptide. These cells may be cultured in a suitable tissue culture medium. However, it is preferable to culture the cells in an Eagle medium (modified Earle) supplemented with 10% fetal bovine serum (inactivated at 56° C.) and supplemented with about 10 g/l of nonessential amino acids, 1000 U/mI of penicillin and about 100 μg/ml of streptomycin.

[0654] The splenocytes of these mice are extracted and fused with a suitable myeloma cell line. However, it is preferable to use the parental myeloma cell line (SP20) available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium and then cloned by limiting dilution as described by Wands et al. (1981, Gastroenterology, 80:225-232). The hybridoma cells obtained after such a selection are tested in order to identify the clones secreting antibodies capable of binding to the polypeptide.

[0655] Moreover, other antibodies capable of binding to the polypeptide may be produced according to a 2-stage procedure using anti-idiotype antibodies such a method is based on the fact that the antibodies are themselves antigens and consequently it is possible to obtain an antibody recognizing another antibody. According to this method, the antibodies specific for the protein are used to immunize an animal, preferably a mouse. The splenocytes of this animal are then used to produce hybridoma cells, and the latter are screened in order to identify the clones which produce an antibody whose capacity to bind to the specific antibody-protein complex may be blocked by the polypeptide. These antibodies may be used to immunize an animal in order to induce the formation of antibodies specific for the protein in a large quantity.

[0656] It is preferable to use Fab and F(ab′)2 and the other fragments of the antibodies of the present invention according to the methods described here. Such fragments are typically produced by proteolytic cleavage with the aid of enzymes such as Papafn (in order to produce the Fab fragments) or Pepsin (in order to produce the F(ab′)2 fragments). Otherwise, the secreted fragments recognizing the protein may be produced by applying the recombinant DNA or synthetic chemistry technology.

[0657] For the in vivo use of antibodies in humans, it would be preferable to use “humanized” chimeric monoclonal antibodies. Such antibodies may be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. The methods for producing the chimeric antibodies are known to persons skilled in the art (for a review, see : Morrison (1985. Science 229:1202); Oi et al., (1986, Biotechnique, 4:214); Cabilly et al., U.S. Pat. No. 4,816,567 ; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al; (1984, Nature, 312:643); and Neuberger et al., (1985, Nature, 314:268).

Example 7 Determination of Polymorphisms/mutations in the ABCA5, ABCA6, ABCA9, or ABCA10 Genes

[0658] The detection of polymorphisms or of mutations in the sequences of the transcripts or in the genomic sequence of the ABCA5, ABCA6, ABCA9, or ABCA10 genes may be carried out according to various protocols. The preferred method is direct sequencing.

[0659] For patients from whom it is possible to obtain an mRNA preparation, the preferred method consists in preparing the cDNAs and sequencing them directly. For patients for whom only DNA is available, and in the case of a transcript where the structure of the corresponding gene is unknown or partially known, it is necessary to precisely determine its intron-exon structure as well as the genomic sequence of the corresponding gene. This therefore involves, in a first instance, isolating the genomic DNA BAC or cosmid clone(s) corresponding to the transcript studied, sequencing the insert of the corresponding clone(s) and detemrining the intron-exon structure by comparing the cDNA sequence to that of the genomic DNA obtained.

[0660] The technique of detection of mutations by direct sequencing consists in comparing the genomic sequences of the ABCA5, ABCA6, ABCA9, or ABCA10 gene obtained from homozygotes for the disease or from at least 8 individuals (4 individuals affected by the pathology studied and 4 individuals not affected) or from at least 32 unrelated individuals from the studied population. The sequence divergences constitute polymorphisms. All those modifying the amino acid sequence of the wild-type protein may be mutations capable of affecting the function of said protein which it is preferred to consider more particularly for the study of cosegregation of the mutation and of the disease (denoted genotype-phenotype correlation) in the pedigree, or of a pharmacological response to a therapeutic molecule in the pharmacogenomic studies, or in the studies of caselcontrol association for the analysis of the sporadic cases.

Example 8 Identification of a Causal Gene for a Disease Linked to a Deficiency in the Transport of Cholesterol and Inflammatory Lipid Substances by Causal Mutation or a Transcriptional Difference

[0661] Among the mutations identified according to the method described in Example 7, all those associated with the disease phenotype are capable of being causal. Validation of these results is made by sequencing the gene in all the affected individuals and their relations (whose DNA is available).

[0662] Moreover, Northern blot or RT-PCR analysis, according to the methods described in Example 2, using RNA specific to affected or nonaffected individuals makes it possible to detect notable variations in the level of expression of the gene studied, in particular in the absence of transcription of the gene.

Example 9 Construction of Recombinant Vectors Comprising a Nucleic Acid Encoding Any One of ABCA5, ABCA6, ABCA9, and ABCA10 Proteins

[0663] Synthesis of a Nucleic Acid Encoding Any One of Human ABCA5, ABCA6. ABCA9, or ABCA10 Proteins

[0664] Total RNA (500 ng) isolated from a human cell (for example, placental tissue, Clontech, Palo Alto, Calif., USA, or THP1 cells) may be used as source for the synthesis of the cDNA of the human ABCA5, ABCA6, ABCA9, and ABCA10 genes. Methods to reverse transcribe mRNA to cDNA are well known in the art. For example, one may use the system “Superscript one step RT-PCR (Life Technologies, Gaithersburg, Md., USA).

[0665] Oligonucleotide primers specific for ABCA5, ABCA6, ABCA9, and ABCA10 cDNAs may be used for this purpose, containing sequences as set forth in any of SEQ ID NOS: 127-217. These oligonucleotide primers may be synthesized by the phosphoramidite method on a DNA synthesizer of the ABI 394 type (Applied Biosystems, Foster City, Calif., USA).

[0666] Sites recognized by the restriction enzyme NotI may be incorporated into the amplified ABCA5, ABCA6, ABCA9, and ABCA10 cDNAs to flank the cDNA region desired for insertion into the recombinant vector by a second amplification step using 50 ng of human ABCA5, ABCA6, ABCA9, and ABCA10 cDNAs as template, and 0.25 μM of the ABCA5, ABCA6, ABCA9, and ABCA10 specific oligonucleotide primers used above containing, at their 5′ end, the site recognized by the restriction enzyme NotI (5′-GCGGCCGC-3′), in the presence of 200 μM of each of said dideoxynucleotides dATP, dCTP, dTTP and dGTP as well as the Pyrococcus furiosus DNA polymerase (Stratagene, Inc. La Jolla, Calif., USA).

[0667] The PCR reaction may be carried out over 30 cycles each comprising a step of denaturation at 95° C. for one minute, a step of renaturation at 50° C. for one minute and a step of extension at 72° C. for two minutes, in a thermocycler apparatus for PCR (Cetus Perkin Elmer Norwalk, Conn., USA).

[0668] Cloning of the cDNA of the Human ABCA5, ABCA6, ABCA9, and ABCA10 Genes Into an Expression Vector

[0669] The human ABCA5, ABCA6, ABCA9, and ABCA10 cDNA inserts may then be cloned into the NotI restriction site of an expression vector, for example, the pCMV vector containing a cytomegalovirus (CMV) early promoter and an enhancer sequence as well as the SV40 polyadenylation signal (Beg et al., 1990, PNAS, 87:3473; Applebaum-Boden, 1996, JCI 97), in order to produce an expression vector designated pABCA5, pABCA6, pABCA9, and pABCA10.

[0670] The sequence of the cloned cDNA can be confirmed by sequencing on the two strands using the reaction set “ABI Prism Big Dye Terminator Cycle Sequencing ready” (marketed by Applied Biosystems, Foster City, Calif., USA) in a capillary sequencer of the ABI 310 type (Applied Biosystems, Foster City, Calif., USA).

[0671] Construction of a Recombinant Adenoviral Vector Containing the cDNA of the Human ABCA5, ABCA6, ABCA9, and ABCA10 Genes

[0672] Modification of the expression vector pCMV-β:

[0673] The β-galactosidase cDNA of the expression vector pCMV-β (Clontech, Palo Alto, Calif., USA, Gene Bank Accession No. UO2451) may be deleted by digestion with the restriction endonuclease NotI and replaced with a multiple cloning site containing, from the 5′ end to the 3′ end, the following sites: NotI, AscI, RsrII, AvrII, SwaI, and NotI, cloned at the region of the NotI restriction site. The sequence of this multiple cloning site is:

[0674] 5′-CGGCCGCGGCGCGCCCGGACCGCCTAGGATTTAAATCGCGGCCCGCG-3′.

[0675] The DNA fragment between the EcoRI and SanI sites of the modified expression vector pCMV may be isolated and cloned into the modified Xbal site of the shuttle vector PXCXII (McKinnon et al., 1982, Gene, 19:33; McGrory et al., 1988, Virology, 163:614).

[0676] Modification of the shuttle vector pXCXII:

[0677] A multiple cloning site comprising, from the 5′ end to the 3 end the XbaI, EcoRI, SfiI, PmeI, NheI, SrfI, PacI, SalI and XbaI restriction sites having the sequence:

[0678] 5′CTCTAGAATTCGGCCTCCGTGGCCGTTTAAACGCTAGCGCCCGGGCTTAATTAAGTCGACTCTAGAGC-3′, may be inserted at the level of the XbaI site (nucleotide at position 3329) of the vector pXCXII (McKinnon et al., 1982, Gene 19:33; McGrory et al., 1988, Virology, 163:614).

[0679] The EcoRI-SalI DNA fragment isolated from the modified vector pCMV-β containing the CMV promoter/enhancer, the donor and acceptor splicing sites of FV40 and the polyadenylation signal of FV40 may then be cloned into the EcoRI-SalI site of the modified shuttle vector pXCX, designated pCMV-11.

[0680] Preparation of the Shuttle Vector pAD12-ABCA

[0681] The human ABCA5, ABCA6, ABCA9, and ABCA10 cDNAs are obtained by an RT-PCR reaction, as described above, and cloned at the level of the NotI site into the vector pCMV-12, resulting in the obtaining of the vector pCMV-ABCA5, pCMV-ABCA6, pCMV-ABCA9, and pCMV-ABCA10.

[0682] Construction of the ABC1 Recombinant Adenovirus

[0683] The recombinant adenovirus containing the human ABCA5, ABCA6, ABCA9, and ABCA10 cDNAs may be constructed according to the technique described by McGrory et al. (1988, Virology, 163:614).

[0684] Briefly, the vector pAD12-ABCA is cotransfected with the vector tGM17 according to the technique of Chen and Okayama (1987, Mol Cell Biol., 7:2745-2752).

[0685] Likewise, the vector pAD12-Luciferase was constructed and cotransfected with the vector pJM17.

[0686] The recombinant adenoviruses are identified by PCR amplification and subjected to two purification cycles before a large-scale amplification in the human embryonic kidney cell line HEK 293 (American Type Culture Collection, Rockville, Md., USA).

[0687] The infected cells are collected 48 to 72 hours after their infection with the adenoviral vectors and subjected to five freeze-thaw lysing cycles.

[0688] The crude lysates are extracted with the aid of Freon (Halocarbone 113, Matheson Product, Scaucus, N.J. USA), sedimented twice in cesium chloride supplemented with 0.2% murine albumine (Sigma Chemical Co., St Louis, Mo., USA) and dialysed extensively against buffer composed of 150 nM NaCl, 10 mM Hepes (pH 7,4), 5 mM KCl, 1 mM MgCl₂, and 1 mM CaCl₂.

[0689] The recombinant adenoviruses are stored at −70° C. and titrated before their administration to animals or their incubation with cells in culture.

[0690] The absence of wild-type contaminating adenovirus is confirmed by screening with the aid of PCR amplification using oligonucleotide primers located in the structural portion of the deleted region.

[0691] Validation of the Expression of the Human ABCA5, ABCA6, ABCA9, and ABCA10 cDNAs

[0692] Polyclonal antibodies specific for a human ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides may be prepared as described above in rabbits and chicks by injecting a synthetic polypeptide fragment derived from an ABC1 protein, comprising all or part of an amino acid sequence as described in SEQ ID NOS:5-8. These polyclonal antibodies are used to detect and/or quantify the expression of the human ABCA5, ABCA6, ABCA9, and ABCA10 genes in cells and animal models by immunoblotting and/or immunodetection.

[0693] The biological activity of ABCA5-6, 9-10 may be monitored by quantifying the cholesterol fluxes induced by apoA-I using cells transfected with the vector pCMV-ABCI which have been loaded with cholesterol (Remaley et al., 1997, ATVB, 17:1813).

[0694] Expression in Vitro of the Human ABCA5, ABCA6, ABCA9, and ABCA10 cDNAs in Cells

[0695] Cells of the HEK293 line and of the COS-7 line (American Tissue Culture Collection, Bethesda, Md., USA), as well as fibroblasts in primary culture derived from Tangier patients or from patients suffering from hypo-alphalipoproteinemia are transfected with the expression vector pCMV-ABCA5, pCMV-ABCA6, pCMV-ABCA9, and pCMV-ABCA10 (5-25 μg) using Lipofectamine (BRL, Gaithersburg, Md., USA) or by coprecipitation with the aid of calcium chloride (Chen et al., 1987, Mol Cell Biol., 7:2745-2752).

[0696] These cells may also be infected with the vector pABCA5-AdV, pABCA6-AdV, pABCA9-AdV, and pABCA10-AdV (Index of infection, MOI=10).

[0697] The expression of human ABCA5-6, 9-10 may be monitored by immunoblotting as well as by quantification of the efflux of cholesterol induced by apoA-1 using transfected and/or infected cells.

[0698] Expression in Vivo of the ABCA5, ABCA6, ABCA9, and ABCA10 Genes in Various Animal Models

[0699] An appropriate volume (100 to 300 μl) of a medium containing the purified recombinant adenovirus (pABCA-AdV or pLucif-AdV) containing from 10⁸ to 10⁹ lysis plaque-forming units (pfu) are infused into the Saphenous vein of mice (C57BL/6, both control mice and models of transgenic or knock-out mice) on day 0 of the experiment.

[0700] The evaluation of the physiological role of the ABCA5, ABCA6, ABCA9, and ABCA10 proteins in the transport of cholesterol or inflammatory lipid substances is carried out by determining the total quantity of cholesterol or appropriate inflammatory lipid substances before (day zero) and after (days 2, 4, 7, 10, 14) the administration of the adenovirus.

[0701] Kinetic studies with the aid of radioactively labelled products are carried out on day 5 after the administration of the vectors rLucif-AdV and rABCA-AdV in order to evaluate the effect of the expression of ABCA5, ABCA6, ABCA9, and ABCA10 on the transport of cholesterol and inflammatory lipid substances.

[0702] Furthermore, transgenic mice and rabbits overexpressing the ABCA5, ABCA6, ABCA9, and ABCA10 genes may be produced, in accordance with the teaching of Vaisman (1995) and Hoeg (1996) using constructs containing the human ABCA5, ABCA6, ABCA9, and ABCA10 cDNAs under the control of endogenous promoters such as ABCA5, ABCA6, ABCA9, and ABCA10, or CMV or apoE.

[0703] The evaluation of the long-term effect of the expression of ABCA5, ABCA6, ABCA9, and ABCA10 on the kinetics of the lipids involved in the mediation of the inflammation may be carried out as described above.

Example 10 Isotopic in Situ Hybridization Study of the ABCA9 Gene

[0704] In situ hybridization experiments were performed using a radiolabeled cRNA probe of 330 bp corresponding to nucleotides 1 to 330 of nucleotide sequence of SEQ ID NO: 3. The 330 bp insert was subcloned and transcribed in vitro with T7 (antisense) and SP6 (sense) RNA polymerases in the presence of ³⁵S-uridine 5′-triphosphate. After transcription, the probes were column-purified and separated by electrophoresis on a 5% polyacrylamide gel to confirm size and purity.

[0705] Serial artery and heart tissue sections were digested with Proteinase K and hybridized with the probes at a concentration of approximately 3.4×10⁷ dpm/ml for 18 hours at 55° C. Following hybridization, the slides were treated with RNAse A and washed stringently in 0.1×SSC at 60° C. for 2 hours. The slides were then coated with Kodak NTB-2 emulsion, exposed for 14 days at 4° C., and developed using Kodak D-19 Developer and Fixer. Slides were stained with hematoxylin and eosin (H&E) and imaged using a DVC 1310C camera coupled to a Nikon microscope.

[0706] Two control probes were used in these studies. All tissues were screened initially with a probe for beta-actin mRNA to ensure that RNAs were preserved within the archival paraffin samples. Adjacent serial sections were also hybridized with a sense control riboprobe that was derived from the same region of the gene as the antisense probe. At these hybridization and wash stringencies, the sense control probe tended to produce a background signal across all tissues that was not associated with particular cell types and that appeared to be due to nonspecific sticking of the sense probe to the tissues. The nonspecific background produced by the antisense probe was less than that observed with the sense probe, and the positive antisense signals described in the accompanying report were specifically cell-associated and higher than the background signals present in nonreactive cell types.

[0707]FIG. 5 was a section of normal renal artery obtained at nephrectomy from an 80-year-old female with renal cell carcinoma, and showed a faint hybridization of medial smooth muscle in both arteries and veins. Adventitial nerves showed occasional faint positivity in Schwann cells (FIG. 6). Also, in an adjacent ganglion, ganglion cells and Schwann cells were both faintly positive (FIG. 7).

[0708]FIGS. 8 and 9 display sections of normal renal artery obtained at nephrectomy from a 24-year-old male with congenital stenosis of the ureteropelvic junction, and show hybridization in a collecting duct epithelium and a renal tubular epithelium, respectively.

[0709]FIG. 10 was a section of a normal heart obtained from a 41-year-old female who died of carcinoma of the cervix, and showed a moderately positive hybridization of the cardiac myocytes.

[0710]FIG. 11 was a section of a normal heart obtained from a 60-year-old male who died of non-small lung carcinoma, and showed that endothelium was occasionally positive in interstitial vessels.

Example 11 Isotopic in Situ Hybridization Study of the ABCA10 Gene

[0711] In situ hybridization experiments have been performed using serial arterial, myocardial, and skeletal tissue sections from archival paraffin samples.

[0712] Tissue sections were hybridized with radiolabeled cRNA probes of 405 bp corresponding to nucleotides 1383 to 1787 of nucleotide sequence SEQ ID NO: 4, which was then PCR-amplified. The PCR product was then transcribed in vitro with T7 (antisense) and SP6 (sense) RNA polymerases in the presence of ³⁵S-uridine 5′-triphosphate. After transcription, the probes were column-purified and separated by electrophoresis on a 5% polyacrylamide gel to confirm size and purity. Tissue sections were digested, and in situ hybridization were perforned as described in Example 10.

[0713]FIGS. 12 and 13 which displays in situ hybridization of arterial tissues showed that the strongest hybridization was identified consistently in macrophages, subsets of lymphocytes, and in Schwann cells of nerves.

[0714]FIG. 14 which display a myocardial tissue section, showed positive signals of macrophages in the atheroma. In an adjacent section of tissue containing a ganglion, subsets of Schwann cells were moderately positive, and ganglion cells showed faint hybridization (FIG. 15).

[0715]FIG. 16 displays section of skeletal tissue, wherein scattered macrophages were faintly to moderately positive. FIG. 17 showed moderate hybridization of Schwann cells in a nerve.

[0716] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

1 217 1 6525 DNA Homo sapiens unsure 4449 n=unknown, may be a or g or c or t 1 aaaatgttga tattttctct tagcaggctg tcaaccaggt taggttcagg tcataagttt 60 ctacccacat tctttgaact gtagttgtca ttttagttta tttttcaaaa acttttgcag 120 tacctttttg gtctgtcttg tgtgtgcctt gcagtgaaca gtctggattt ggacagtggt 180 ctgtctgtta gttcagtttc tcaagccttt gtcacactaa taggattgga tttatgtatg 240 tccagcttgg gaattattac aggaattaaa aacaactttt tagagtgctt tcctgagctc 300 tctttctatt tgttccccct tctacttttt gcttccctgt ggctgctgtt tctatcctcc 360 agccagagag ctagtgttta ttttctccat tgtgttacac acttgtgcag ctgcaaccac 420 catatccagg gcccaatggt aggaggtaga gaagaaaagc aaaagggatt ggcctcatcc 480 tcttacaacg atagttccat tgaatagaga gaaaggtttt cctgcctcag agtgttggct 540 gcactaggct tttgttactg tagtctggcc ctgttaccat gggattgctt gcatgtgggg 600 atacaggaga attcagaaaa gaaaaaaaga tttgctattt ctacattctc cctgagcatt 660 aagacctccc ttgcccattc ctcaattcaa agctaaggct tcttctggag ctgcctctgt 720 gggcggttcg ggagatacca aaggagaaaa agtaccactg ttgatatggt ggtatttcaa 780 attctggtct accctatttc acatgccttg tttacttttc agagctgaca gattgctgct 840 ccatgcattc tgtccagttt cctaagagag acagcttgga gtatgcttaa tccatcttac 900 ctgggactga aacagctgct tattttgccg ttaaaaatta catgcagttt actgcgtggc 960 tccgggtttg tttgtttgtt tttcctcttt aataggttta ttcagaaaac atgtccactg 1020 caattaggga ggtaggagtt tggagacaga ccagaacact tctactgaag aattacttaa 1080 ttaaatgcag aaccaaaaag agtagtgttc aggaaattct ttttccacta ttttttttat 1140 tttggttaat attaattagc atgatgcatc caaataagaa atatgaagaa gtgcctaata 1200 tagaactcaa tcctatggac aagtttactc tttctaatct aattcttgga tatactccag 1260 tgactaatat tacaagcagc atcatgcaga aagtgtctac tgatcatcta cctgatgtca 1320 taattactga agaatataca aatgaaaaag aaatgttaac atccagtctc tctaagccga 1380 gcaactttgt aggtgtggtt ttcaaagact ccatgtccta tgaacttcgt ttttttcctg 1440 atatgattcc agtatcttct atttatatgg attcaagagc tggctgttca aaatcatgtg 1500 aggctgctca gtactggtcc tcaggtttca cagttttaca agcatccata gatgctgcca 1560 ttatacagtt gaagaccaat gtttctcttt ggaaggagct ggagtcaact aaagctgtta 1620 ttatgggaga aactgctgtt gtagaaatag atacctttcc ccgaggagta attttaatat 1680 acctagttat agcattttca ccttttggat actttttggc aattcatatc gtagcagaaa 1740 aagaaaaaaa aataaaagaa tttttaaaga taatgggact tcatgatact gccttttggc 1800 tttcctgggt tcttctatat acaagtttaa tttttcttat gtcccttctt atggcagtca 1860 ttgcgacagc ttctttgtta tttcctcaaa gtagcagcat tgtgatattt ctgctttttt 1920 tcctttatgg attatcatct gtattttttg ctttaatgct gacacctctt tttaaaaaat 1980 caaaacatgt gggaatagtt gaattttttg ttactgtggc ttttggattt attggcctta 2040 tgataatcct catagaaagt tttcccaaat cgttagtgtg gcttttcagt cctttctgtc 2100 actgtacttt tgtgattggt attgcacagg tcatgcattt agaagatttt aatgaaggtg 2160 cttcattttc aaatttgact gcaggcccat atcctctaat tattacaatt atcatgctca 2220 cacttaatag tatattctat gtcctcttgg ctgtctatct tgatcaagtc attccagggg 2280 aatttggctt acggagatca tctttatatt ttctgaagcc ttcatattgg tcaaagagta 2340 aaagaaatta tgaggagtta tcagagggca atgttaatgg aaatattagt tttagtgaaa 2400 ttattgagcc agtttcttca gaatttgtag gaaaagaagc cataagaatt agtggtattc 2460 agaagacata cagaaagaag ggtgaaaatg tggaggcttt gagaaatttg tcatttgaca 2520 tatatgaggg tcagattact gccttacttg gccacagtgg aacaggaaag agtacattga 2580 tgaatattct ttgtggactc tgcccacctt ctgatgggtt tgcatctata tatggacaca 2640 gagtctcaga aatagatgaa atgtttgaag caagaaaaat gattggcatt tgtccacagt 2700 tagatataca ctttgatgtt ttgacagtag aagaaaattt atcaattttg gcttcaatca 2760 aagggatacc agccaacaat ataatacaag aagtgcagaa ggttttacta gatttagaca 2820 tgcagactat caaagataac caagctaaaa aattaagtgg tggtcaaaaa agaaagctgt 2880 cattaggaat tgctgttctt gggaacccaa agatactgct gctagatgaa ccaacagctg 2940 gaatggaccc ctgttctcga catattgtat ggaatctttt aaaatacaga aaagccaatc 3000 gggtgacagt gttcagtact catttcatgg atgaagctga cattcttgca gataggaaag 3060 ctgtgatatc acaaggaatg ctgaaatgtg ttggttcttc aatgttcctc aaaagtaaat 3120 gggggatcgg ctaccgcctg agcatgtaca tagacaaata ttgtgccaca gaatctcttt 3180 cttcactggt taaacaacat atacctggag ctactttatt acaacagaat gaccaacaac 3240 ttgtgtatag cttgcctttc aaggacatgg acaaattttc aggtttgttt tctgccctag 3300 acagtcattc aaatttgggt gtcatttctt atggtgtttc catgacgact ttggaagacg 3360 tatttttaaa gctagaagtt gaagcagaaa ttgaccaagc agattatagt gtatttactc 3420 agcagccact ggaggaagaa atggattcaa aatcttttga tgaaatggaa cagagcttac 3480 ttattctttc tgaaaccaag gcttctctag tgagcaccat gagcctttgg aaacaacaga 3540 tgtatacaat agcaaagttt catttcttta ccttgaaacg tgaaagtaaa tcagtgagat 3600 cagtgttgct tctgctttta atttttttca cagttcagat ttttatgttt ttggttcatc 3660 actcttttaa aaatgctgtg gttcccatca aacttgttcc agacttatat tttctaaaac 3720 ctggagacaa accacataaa tacaaaacaa gtctgcttct tcaaaattct gctgactcag 3780 atatcagtga tcttattagc tttttcacaa gccagaacat aatggtgacg atgattaatg 3840 acagtgacta tgtatccgtg gctccccata gtgcggcttt aaatgtgatg cattcagaaa 3900 aggactatgt ttttgcagct gttttcaaca gtactatggt ttattcttta cctatattag 3960 tgaatatcat tagtaactac tatctttatc atttaaatgt gactgaaacc atccagatct 4020 ggagtacccc attctttcaa gaaattactg atatagtttt taaaattgag ctgtattttc 4080 aagcagcttt gcttggaatc attgttactg caatgccacc ttactttgcc atggaaaatg 4140 cagagaatca taagatcaaa gcttatactc aacttaaact ttcaggtctt ttgccatctg 4200 catattggat tggacaagct gttgttgata tccccttatt ttttatcatt cttattttga 4260 tgctaggaag cttactggca tttcattatg gattatattt ttatactgta aagttccttg 4320 ctgtggtttt ttgccttatt ggttatgttc catcagttat tctgttcact tatattgctt 4380 ctttcacctt taagaaaatt ttaaatacca aagaattttg gtcatttatc tattctgtgg 4440 cagcgttgnc ttgtattgca atcactgaaa taactttctt tatgggatac acaattgcaa 4500 ctattcttca ttatgccttt tgtatcatca ttccaatcta tccacttcta ggttgcctga 4560 tttctttcat aaagatttct tggaagaatg tacgaaaaaa tgtggacacc tataatccat 4620 gggataggct ttcagtagct gttatatcgc cttacctgca gtgtgtactg tggattttcc 4680 tcttacaata ctatgagaaa aaatatggag gcagatcaat aagaaaagat ccctttttca 4740 gaaacctttc aacgaagtct aaaaatagga agcttccaga accaccagac aatgaggatg 4800 aagatgaaga tgtcaaagct gaaagactaa aggtcaaaga gctgatgggt tgccagtgtt 4860 gtgaggagaa accatccatt atggtcagca atttgcataa agaatatgat gacaagaaag 4920 attttcttct ttcaagaaaa gtaaagaaag tggcaactaa atacatctct ttctgtgtga 4980 aaaaaggaga gatcttagga ctattgggtc caaatggtgc tggcaaaagc acaattatta 5040 atattctggt tggtgatatt gaaccaactt caggccaggt atttttagga gattattctt 5100 cagagacaag tgaagatgat gattcactga agtgtatggg ttactgtcct cagataaacc 5160 ctttgtggcc agatactaca ttgcaggaac attttgaaat ttatggagct gtcaaaggaa 5220 tgagtgcaag tgacatgaaa gaagtcataa gtcgaataac acatgcactt gatttaaaag 5280 aacatcttca gaagactgta aagaaactac ctgcaggaat caaacgaaag ttgtgttttg 5340 ctctaagtat gctagggaat cctcagatta ctttgctaga tgaaccatct acaggtatgg 5400 atcccaaagc caaacagcac atgtggcgag caattcgaac tgcatttaaa aacagaaagc 5460 gggctgctat tctgaccact cactatatgg aggaggcaga ggctgtctgt gatcgagtag 5520 ctatcatggt gtctgggcag ttaagatgta tcggaacagt acaacatcta aagagtaaat 5580 ttggaaaagg ctactttttg gaaattaaat tgaaggactg gatagaaaac ctagaagtag 5640 accgccttca aagagaaatt cagtatattt tcccaaatgc aagccgtcag gaaagttttt 5700 cttctatttt ggcttataaa attcctaagg aagatgttca gtccctttca caatcttttt 5760 ttaagctgga agaagctaaa catgcttttg ccattgaaga atatagcttt tctcaagcaa 5820 cattggaaca ggtttttgta gaactcacta aagaacaaga ggaggaagat aatagttgtg 5880 gaactttaaa cagcacactt tggtgggaac gaacacaaga agatagagta gtattttgaa 5940 tttgtattgt tcggtctgct tactgggact tctttctttt tcacttaatt ttaactttgg 6000 tttaaaaagt tttttattgg aatggtaact ggagaaccaa gaacgcactt gaaatttttc 6060 taagctcctt aattgaaatg ctgtggttgt gtgttttgct tttctttaaa taaaacgtat 6120 gtataattaa gtgaagctgc atgtttgtat tgaagtatat tgaactatat agtttgtatg 6180 tcatcttttt caccattcag aaacagtgct tctgaatttg tgatttaaag gaattgtaat 6240 agaatagttt tatttttaag ttatctttaa gtttatgcca tcttcttaaa taagtacgta 6300 atgttccaat ctaaataaaa aactaataca taactaatgc atagaaaaga tacataaagc 6360 aatgtgaaag tttcttgctt ctccttttta atttctaaaa aagccacttt gaatggaagt 6420 tgtcatccgt aaaagctgaa gtgtaagcac taggaaatct caatatagag atttgaggaa 6480 agttatatcc actaggtggc agtcattgat cataataagt gaaat 6525 2 5296 DNA Homo sapiens 2 ctgctggagt aggcacccat ttaaagaaaa aatgaagaag cagcaataaa gaagttgtaa 60 tcgttaccta gacaaacaga gaactggttt tgacagtgtt tctagagtgc tttttattat 120 tttcctgaca gttgtgttcc accatgatta ctttctcctt cagcgaatag gctaaatgaa 180 tatgaaacag aaaagcgtgt atcagcaaac caaagcactt ctgtgcaaga attttcttaa 240 gaaatggagg atgaaaagag agagcttatt ggaatggggc ctctcaatac ttctaggact 300 gtgtattgct ctgttttcca gttccatgag aaatgtccag tttcctggaa tggctcctca 360 gaatctggga agggtagata aatttaatag ctcttcttta atggttgtgt atacaccaat 420 atctaattta acccagcaga taatgaataa aacagcactt gctcctcttt tgaaaggaac 480 aagtgtcatt ggggcaccaa ataaaacaca catggacgaa atacttctgg aaaatttacc 540 atatgctatg ggaatcatct ttaatgaaac tttctcttat aagttaatat ttttccaggg 600 atataacagt ccactttgga aagaagattt ctcagctcat tgctgggatg gatatggtga 660 gttttcatgt acattgacca aatactggaa tagaggattt gtggctttac aaacagctat 720 taatactgcc attatagaaa tcacaaccaa tcaccctgtg atggaggagt tgatgtcagt 780 tactgctata actatgaaga cattaccttt cataactaaa aatcttcttc acaatgagat 840 gtttatttta ttcttcttgc ttcatttctc cccacttgta tattttatat cactcaatgt 900 aacaaaagag agaaaaaagt ctaagaattt gatgaaaatg atgggtctcc aagattcagc 960 attctggctc tcctggggtc taatctatgc tggcttcatc tttattattt ccatattcat 1020 tacaattatc ataacattca cccaaattat agtcatgact ggcttcatgg tcatatttat 1080 actctttttt ttatatggct tatctttggt agctttggtg ttcctgatga gtgtgctgtt 1140 aaagaaagct gtcctcacca atttggttgt gtttctcctt accctctttt ggggatgtct 1200 gggattcact gtattttatg aacaacttcc ttcatctctg gagtggattt tgaatatttg 1260 tagccctttt gcctttacta ctggaatgat tcagattatc aaactggatt ataacttgaa 1320 tggtgtaatt tttcctgacc cttcaggaga ctcatataca atgatagcaa ctttttctat 1380 gttgcttttg gatggtctca tctacttgct attggcatta tactttgaca aaattttacc 1440 ctatggagat gagcgccatt attctccttt atttttcttg aattcatcat cttgtttcca 1500 acaccaaagg actaatgcta aggttattga gaaagaaatc gatgctgagc atccctctga 1560 tgattatttt gaaccagtag ctcctgaatt ccaaggaaaa gaagccatca gaatcagaaa 1620 tgttaagaag gaatataaag gaaaatctgg aaaagtggaa gcattgaaag gcttgctctt 1680 tgacatatat gaaggtcaaa tcacggcaat cctgggtcac agtggagctg gcaaatcttc 1740 actgctaaat attcttaatg gattgtctgt tccaacagaa ggatcagtta ccatctataa 1800 taaaaatctc tctgaaatgc aagacttgga ggaaatcaga aagataactg gcgtctgtcc 1860 tcaattcaat gttcaatttg acatactcac cgtgaaggaa aacctcagcc tgtttgctaa 1920 aataaaaggg attcatctaa aggaagtgga acaagaggta caacgaatat tattggaatt 1980 ggacatgcaa aacattcaag ataaccttgc taaacattta agtgaaggac agaaaagaaa 2040 gctgactttt gggattacca ttttaggaga tcctcaaatt ttgcttttag atgaaccaac 2100 tactggattg gatccctttt ccagagatca agtgtggagc ctcctgagag agcgtagagc 2160 agatcatgtg atccttttca gtacccagtc catggatgag gctgacatcc tggctgatag 2220 aaaagtgatc atgtccaatg ggagactgaa gtgtgcaggt tcttctatgt ttttgaaaag 2280 aaggtggggt cttggatatc acctaagttt acataggaat gaaatatgta acccagaaca 2340 aataacatcc ttcattactc atcacatccc cgatgctaaa ttaaaaacag aaaacaaaga 2400 aaagcttgta tatactttgc cactggaaag gacaaataca tttccagatc ttttcagtga 2460 tctggataag tgttctgacc agggagtgac aggttatgac atttccatgt caactctaaa 2520 tgaagtcttt atgaaactgg aaggacagtc aactatcgaa caagatttcg aacaagtgga 2580 gatgataaga gactcagaaa gcctcaatga aatggagctg gctcactctt ccttctctga 2640 aatgcagaca gctgtgagtg acatgggcct ctggagaatg caagtctttg ccatggcacg 2700 gctccgtttc ttaaagttaa aacgtcaaac taaagtgtta ttgaccctat tattggtatt 2760 tggaatcgca atattccctt tgattgttga aaatataata tatgctatgt taaatgaaaa 2820 gatcgattgg gaatttaaaa acgaattgta ttttctctct cctggacaac ttccccagga 2880 accccgtacc agcctgttga tcatcaataa cacagaatca aatattgaag attttataaa 2940 atcactgaag catcaaaata tacttttgga agtagatgac tttgaaaaca gaaatggtac 3000 tgatggcctc tcatacaatg gagctatcat agtttctggt aaacaaaagg attatagatt 3060 ttcagttgtg tgtaatacca agagattgca ctgttttcca attcttatga atattatcag 3120 caatgggcta cttcaaatgt ttaatcacac acaacatatt cgaattgagt caagcccatt 3180 tcctcttagc cacataggac tctggactgg gttgccggat ggttcctttt tcttattttt 3240 ggttctatgt agcatttctc cttatatcac catgggcagc atcagtgatt acaagaaaaa 3300 tgctaagtcc cagctatgga tttcaggcct ctacacttct gcttactggt gtgggcaggc 3360 actagtggac gtcagcttct tcattttaat tctcctttta atgtatttaa ttttctacat 3420 agaaaacatg cagtaccttc ttattacaag ccaaattgtg tttgctttgg ttatagttac 3480 tcctggttat gcagcttctc ttgtcttctt catatatatg atatcattta tttttcgcaa 3540 aaggagaaaa aacagtggcc tttggtcatt ttacttcttt tttgcctcca ccatcatgtt 3600 ttccatcact ttaatcaatc attttgacct aagtatattg attaccacca tggtattggt 3660 tccttcatat accttgcttg gatttaaaac ttttttggaa gtgagagacc aggagcacta 3720 cagagaattt ccagaggcaa attttgaatt gagtgccact gattttctag tctgcttcat 3780 accctacttt cagactttgc tattcgtttt tgttctaaga tgcatggaac taaaatgtgg 3840 aaagaaaaga atgcgaaaag atcctgtttt cagaatttcc ccccaaagta gagatgctaa 3900 gccaaatcca gaagaaccca tagatgaaga tgaagatatt caaacagaaa gaataagaac 3960 agccactgct ctgaccactt caatcttaga tgagaaacct gttataattg ccagctgtct 4020 acacaaagaa tatgcaggcc agaagaaaag ttgcttttca aagaggaaga agaaaatagc 4080 agcaagaaat atctctttct gtgttcaaga aggtgaaatt ttgggattgc taggacccag 4140 tggtgctgga aaaagttcat ctattagaat gatatctggg atcacaaagc caactgctgg 4200 agaggtggaa ctgaaaggct gcagttcagt tttgggccac ctggggtact gccctcaaga 4260 gaacgtgctg tggcccatgc tgacgttgag ggaacacctg gaggtgtatg ctgccgtcaa 4320 ggggctcagg aaagcggacg cgaggctcgc catcgcaaga ttagtgagtg ctttcaaact 4380 gcatgagcag ctgaatgttc ctgtgcagaa attaacagca ggaatcacga gaaagttgtg 4440 ttttgtgctg agcctcctgg gaaactcacc tgtcttgctc ctggatgaac catctacggg 4500 catagacccc acagggcagc agcaaatgtg gcaggcaatc caggcagtcg ttaaaaacac 4560 agagagaggt gtcctcctga ccacccataa cctggctgag gcggaagcct tgtgtgaccg 4620 tgtggccatc atggtgtctg gaaggcttag atgcattggc tccatccaac acctgaaaaa 4680 caaacttggc aaggattaca ttctagagct aaaagtgaag gaaacgtctc aagtgacttt 4740 ggtccacact gagattctga agcttttccc acaggctgca gggcaggaaa ggtattcctc 4800 tttgttaacc tataagctgc ccgtggcaga cgtttaccct ctatcacaga cctttcacaa 4860 attagaagca gtgaagcata actttaacct ggaagaatac agcctttctc agtgcacact 4920 ggagaaggta ttcttagagc tttctaaaga acaggaagta ggaaattttg atgaagaaat 4980 tgatacaaca atgagatgga aactcctccc tcattcagat gaaccttaaa acctcaaacc 5040 tagtaatttt ttgttgatct cctataaact tatgttttat gtaataatta atagtatgtt 5100 taattttaaa gatcatttaa aattaacatc aggtatattt tgtaaattta gttaacaaat 5160 acataaattt taaaattatt cttcctctca aacatagggg tgatagcaaa cctgtgataa 5220 aggcaataca aaatattagt aaagtcaccc aaagagtcag gcactgggta ttgtggaaat 5280 aaaactatat aaactt 5296 3 5981 DNA Homo sapiens 3 attcacaatg aatgtgaaat taaaagcatg atgtagtagt gacccaaaag gaatgtgaat 60 tctcctccag aacatgcaga gacccatgga tgaactgtgt ttctagattt ttcctccagc 120 tttcctgaga gaaacaggtc aaaatgagca agagacgcat gagcgtgggt cagcaaacat 180 gggctcttct ctgcaagaac tgtctcaaaa aatggagaat gaaaagacag accttgttgg 240 aatggctctt ttcatttctt ctggtactgt ttctgtacct atttttctcc aatttacatc 300 aagttcatga cactcctcaa atgtcttcaa tggatctggg acgtgtagat agttttaatg 360 atactaatta tgttattgca tttgcacctg aatccaaaac tacccaagag ataatgaaca 420 aagtggcttc agccccattc ctaaaaggaa gaacaatcat ggggtggcct gatgaaaaaa 480 gcatggatga attggatttg aactattcaa tagacgcagt gagagtcatc tttactgata 540 ccttctccta ccatttgaag ttttcttggg gacatagaat ccccatgatg aaagagcaca 600 gagaccattc agctcactgt caagcagtga atgaaaaaat gaagtgtgaa ggttcagagt 660 tctgggagaa aggctttgta gcttttcaag ctgccattaa tgctgctatc atagaaatcg 720 caacaaatca ttcagtgatg gaacagctga tgtcagttac tggtgtacat atgaagatat 780 taccttttgt tgcccaagga ggagttgcaa ctgatttttt cattttcttt tgcattattt 840 ctttttctac atttatatac tatgtatcag tcaatgttac acaagaaaga caatacatta 900 cgtcattgat gacaatgatg ggactccgag agtcagcatt ctggctttcc tggggtttga 960 tgtatgctgg cttcatcctt atcatggcca ctttaatggc tcttattgta aaatctgcac 1020 aaattgtcgt cctgactggt tttgtgatgg tcttcaccct ctttctcctc tatggcctgt 1080 ctttgataac tttagctttc ctgatgagtg tgttgataaa gaaacctttc cttacgggct 1140 tggttgtgtt tctccttatt gtcttttggg ggatcctggg attcccagca ttgtatacac 1200 atcttcctgc atttttggaa tggactttgt gtcttcttag cccctttgcc ttcactgttg 1260 ggatggccca gcttatacat ttggactatg atgtgaattc taatgcccac ttggattctt 1320 cacaaaatcc atacctcata atagctactc ttttcatgtt ggtttttgac acccttctgt 1380 atttggtatt gacattatat tttgacaaaa ttttgcccgc tgaatatgga catcgatgtt 1440 ctcccttgtt tttcctgaaa tcctgttttt ggtttcaaca cggaagggct aatcatgtgg 1500 tccttgagaa tgaaacagat tctgatccta cacctaatga ctgttttgaa ccagtgtctc 1560 cagaattctg tgggaaggaa gccatcagaa tcaaaaatct taaaaaagaa tatgcaggga 1620 agtgtgagag agtagaagct ttgaaaggtg tggtgtttga catatatgaa ggccagatca 1680 ctgccctcct tggtcacagt ggagctggaa aaactaccct gttaaacata cttagtgggt 1740 tgtcagttcc aacatcaggt tcagtcactg tctataatca cacactttca agaatggctg 1800 atatagaaaa tatcagcaag ttcactggat tttgtccaca atccaatgtg caatttggat 1860 ttctcactgt gaaagaaaac ctcaggctgt ttgctaaaat aaaagggatt ttgccacatg 1920 aagtggagaa agaggtacaa cgagttgtac aggaattaga aatggaaaat attcaagaca 1980 tccttgctca aaacttaagt ggtggacaaa ataggaaact aacttttggg attgccattt 2040 taggagatcc tcaagttttg ctattggatg aaccgactgc tggattggat cctctttcaa 2100 ggcaccgaat atggaatctc ctgaaagagg ggaaatcaga cagagtaatt ctcttcagca 2160 cccagtttat agatgaggct gacattctgg cggacaggaa ggtgttcata tccaatggga 2220 agctgaagtg tgcaggctct tctctgttcc ttaagaagaa atggggcata ggctaccatt 2280 taagtttgca tctgaatgaa aggtgtgatc cagagagtat aacatcactg gttaagcagc 2340 acatctctga tgccaaattg acagcacaaa gtgaagaaaa acttgtatat attttgcctt 2400 tggaaaggac aaacaaattt ccagaacttt acagggatct tgatagatgt tctaaccaag 2460 gcattgagga ttatggtgtt tccataacaa ctttgaatga ggtgtttctg aaattagaag 2520 gaaaatcaac tattgatgaa tcagatattg gaatttgggg acaattacaa actgatgggg 2580 caaaagatat aggaagcctt gttgagctgg aacaagtttt gtcttccttc cacgaaacaa 2640 ggaaaacaat cagtggcgtg gcgctctgga ggcagcaggt ctgtgcaata gcaaaagttc 2700 gcttcctaaa gttaaagaaa gaaagaaaaa gcctgtggac tatattattg ctttttggta 2760 ttagctttat ccctcaactt ttggaacatc tattctacga gtcatatcag aaaagttacc 2820 cgtgggaact gtctccaaat acatacttcc tctcaccagg acaacaacca caggatcctc 2880 tgacccattt actggtcatc aataagacag ggtcaaccat tgataacttt ttacattcac 2940 tgaggcgaca gaacatagct atagaagtgg atgcctttgg aactagaaat ggcacagatg 3000 acccatctta caatggtgct atcattgtgt caggtgatga aaaggatcac agattttcaa 3060 tagcatgtaa tacaaaacgg ctgaattgct ttcctgtcct cctggatgtc attagcaatg 3120 gactacttgg aatttttaat tcgtcagaac acattcagac tgacagaagc acattttttg 3180 aagagcatat ggattatgag tatgggtacc gaagtaacac cttcttctgg ataccgatgg 3240 cagcctcttt cactccatac attgcaatga gcagcattgg tgactacaaa aaaaaagctc 3300 attcccagct acggatttca ggcctctacc cttctgcata ctggtttggc caagcactgg 3360 tggatgtttc cctgtacttt ttgatcctcc tgctaatgca aataatggat tatattttta 3420 gcccagagga gattatattt ataattcaaa acctgttaat tcaaatcctg tgtagtattg 3480 gctatgtctc atctcttgtt ttcttgacat atgtgatttc attcattttt cgcaatggga 3540 gaaaaaatag tggcatttgg tcatttttct tcttaattgt ggtcatcttc tcgatagttg 3600 ctactgatct aaatgaatat ggatttctag ggctattttt tggcaccatg ttaatacctc 3660 ccttcacatt gattggctct ctattcattt tttctgagat ttctcctgat tccatggatt 3720 acttaggagc ttcagaatct gaaattgtat acctggcact gctaatacct taccttcatt 3780 ttctcatttt tcttttcatt ctgcgatgcc tagaaatgaa ctgcaggaag aaactaatga 3840 gaaaggatcc tgtgttcaga atttctccaa gaagcaacgc tatttttcca aacccagaag 3900 agcctgaagg agaggaggaa gatatccaga tggaaagaat gagaacagtg aatgctatgg 3960 ctgtgcgaga ctttgatgag acacccgtca tcattgccag ctgtctacgg aaggaatatg 4020 caggcaaaaa gaaaaattgc ttttctaaaa ggaagaaaac aattgccaca agaaatgtct 4080 ctttttgtgt taaaaaaggt gaagttatag gactgttagg acacaatgga gctggtaaaa 4140 gtacaactat taagatgata actggagaca caaaaccaac tgcaggacag gtgattttga 4200 aagggagcgg tggaggggaa cccctgggct tcctggggta ctgccctcag gagaatgcgc 4260 tgtggcccaa cctgacagtg aggcagcacc tggaggtgta cgctgccgtg aaaggtctca 4320 ggaaagggga cgcaatgatc gccatcacac ggttagtgga tgcgctcaag ctgcaggacc 4380 agctgaaggc tcccgtgaag accttgtcag agggaataaa gcgaaagctg cgctttgtgc 4440 tgagcatcct ggggaacccg tcagtggtgc ttctggatga gccgtcgacc gggatggacc 4500 ccgaggggca gcagcaaatg tggcaggtga ttcgggccac ctttagaaac acggagaggg 4560 gcgccctcct gaccacccac tacatggcag aggctgaggc ggtgtgtgac cgagtggcca 4620 tcatggtgtc aggaaggctg agatgtattg gttccatcca acacctgaaa agcaaatttg 4680 gcaaagacta cctgctggag atgaagctga agaacctggc acaaatggag cccctccatg 4740 cagagatcct gaggcttttc ccccaggctg ctcagcagga aaggttctcc tccctgatgg 4800 tctataagtt gcctgttgag gatgtgcgac ctttatcaca ggctttcttc aaattagaga 4860 tagttaaaca gagtttcgac ctggaggagt acagcctctc acagtctacc ctggagcagg 4920 ttttcctgga gctctccaag gagcaggagc tgggtgatct tgaagaggac tttgatccct 4980 cggtgaagtg gaaactcctc ctgcaggaag agccttaaag ctccaaatac cctatatctt 5040 tctttaatcc tgtgactctt ttaaagataa tattttatag ccttaatatg ccttatatca 5100 gaggtggtac aaaatgcatt tgaaactcat gcaataatta tcctcagtag tatttcttac 5160 agtgagacaa caggcaatgt cagtgagggc gatcgtaggg cataagccta agccatacca 5220 tgcagccttt gtgccagcaa ccaaatccca tgtttcctac tgtgttaagt ttaaaaatgc 5280 atttattata gaattgtcta catttctgag gatgtcatgg agaatgctta attttctttc 5340 tctgaacttc aaaatattaa atattttctt atttttttga ttaaagtata aattaagaca 5400 ccctattgac ttccgggtaa ggggagtcaa ttgattaccc agcagcacag tatttgcttt 5460 ttataattcc ctttttaaat acttgttctt aattgactgg ttttcctttt ctgtcatttt 5520 tcagagttta gattgtgagt ccatgttttg tctgttgtgc ctataaagga aatttgaaat 5580 ctgtatcatt ctactataaa gacacatgca cacgtatgtt tattgcagca ctgtttacaa 5640 tagcaaagac ttggaaccaa ccaaaatacc cacaaatgat agaccggata aagaaaacgt 5700 gacacatata caccatggaa tactatgcag ccatagaaaa ggatgagttc atattcttca 5760 cagggacatg gatgaagctg gaaaccatca tcctcagcaa actaacacag gaacagaaaa 5820 ccaaacaccg catgttctca ctcataagtg ggaattgaac aatgagaata catggacaca 5880 gggaggggaa caccacaccc tggggcctgt tggggggatg ggggctaggg gagggatagc 5940 attaggagaa atacctgatg tagatgatgg gttgatgggt g 5981 4 6181 DNA Homo sapiens unsure 1420 n=unknown, may be a or g or c or t 4 aattaatttt acttaggata agtgttgtta ttattgtttt tattgttgtt ctgttagtta 60 ctcaaaactt cattctaatt gtgccctgag tttgttaaaa taccatactg tatttttgtg 120 taacatgtaa ataggcatta atttttgaga aatagaaatg tttatcctta atgtattttt 180 aatttgctaa cattgatttt ttattttctt tcctgaaata gcttatttcc taaaatgaaa 240 gaatttattc tcagatgaat aatttttata tcagctattc ttatcagagc aataaacaaa 300 taccaatgat gcgctcagcc aacaattcat tacactctct gaagagtaac tggacaagga 360 gaaaaacata gggaaaaaac caacagaatt tgttggcatg ttctacacac agaccatggc 420 ttttcagaag ccaagctgaa taaaaacagt tttaaaagag gcaaccattt gtagaggagt 480 ccttgaagga ttcttcattg ttttcttgga caaaaagaga ccagtggatc caagtgcttc 540 aaatacttct ctcttatttt cttaactcta ttgctctgca atatttactt taccctgtta 600 atgaacagga caaaatggtt aaaaaagaga taagcgtgcg tcaacaaatt caggctcttc 660 tgtacaagaa ttttcttaaa aaatggagaa taaaaagaga gtttattgga atggacaata 720 acattgtttc tagggctata tttgtgcatc ttttcggaac acttcagagc tacccgtttt 780 cctgaacaac ctcctaaagt cctgggaagc gtggatcagt ttaatgactc tggcctggta 840 gtggcatata caccagtcag taacataaca caaaggataa tgaataagat ggccttggct 900 tcctttatga aaggaagaac agtcattggg acaccagatg aagagaccat ggatatagaa 960 cttccaaaaa aataccatga aatggtggga gttatattta gtgatacttt ctcatatcgc 1020 ctgaagttta attggggata tagaatccca gttataaagg agcactctga atacacagaa 1080 cactgttggg ccatgcatgg tgaaattttt tgttacttgg caaagtactg gctaaaaggg 1140 tttgtagctt ttcaagctgc aattaatgct gcaattatag aagtcacaac aaatcattct 1200 gtaatggagg agttgacatc agttattgga ataaatatga agataccacc tttcatttct 1260 aagggagaaa ttatgaatga atggtttcat tttacttgct tagtttcttt ctcttctttt 1320 atatactttg catcattaaa tgttgcaagg gaaagaggaa aatttaagaa actgatgaca 1380 gtaatgggtc tccgagagtc agcattctgg ctctcctggn gattgacata catttgcttc 1440 atcttcatta tgtccatttt tatggctctg gtcataacat caatctcaat tgtatttcat 1500 actggcttca tggtgatatt cacactctat agcttatatg gcctttcttt gatagcattg 1560 gctttcctca tgagtgtttt aataaggaaa cctatgctcg ctggtttggc tggatttctc 1620 ttcactgtat tttggggatg tctgggattc actgtgttat acagacaact tcctttatct 1680 ttgggatggg tattaagtct tcttagccct tttgccttca ctgctggaat ggcccaggtt 1740 acacacctgg ataattactt aagtggtgtt atttttcctg atccctctgg ggattcatac 1800 aaaatgatag ccactttttt cattttggca tttgatactc ttttctattt gatattcaca 1860 ttatattttg agcgagtttt acctgataaa gatggccatg gggattctcc attatttttc 1920 cttaagtcct cattttggtc caaacatcaa aatactcatc atgaaatctt tgagaatgaa 1980 ataaatcctg agcattcctc tgatgattct tttgaaccgg tgtctccaga attccatgga 2040 aaagaagcca taagaatcag aaatgttata aaagaatata atggaaagac tggaaaagta 2100 gaagcattgc aaggcatatt ttttgacata tatgaaggac agatcactgc aatacttggg 2160 cataatggag ctggtaaatc aacactgcta aacattctta gtggattgtc tgtttctaca 2220 gaaggatcag ccactattta taatactcaa ctctctgaaa taactgacat ggaagaaatt 2280 agaaagaata ttggattttg tccacagttc aattttcaat ttgacttcct cactgtgaga 2340 gaaaacctca gggtatttgc taaaataaaa gggattcagc caaaggaagt ggaacaagag 2400 gtaaaaagaa ttataatgga attagacatg caaagcattc aagacattat tgctaaaaaa 2460 ttaagtggtg ggcagaagag aaaactaaca ctagggattg ccatcttagg agatcctcag 2520 gttttgctgc tagatgaacc aactgctgga ttggatccct tttcaagaca ccgagtgtgg 2580 agcctcctga aggagcataa agtagaccga cttatcctct tcagtaccca attcatggat 2640 gaggctgaca tcttggctga taggaaagta tttctgtcta atgggaagtt gaaatgtgca 2700 ggatcatctt tgtttctgaa gcgaaagtgg ggtattggat atcatttaag tttacacagg 2760 aatgaaatgt gtgacacaga aaaaatcaca tcccttatta agcagcacat tcctgatgcc 2820 aagttaacaa cagaaagtga agaaaaactt gtatatagtt tgcctttgga aaaaacgaac 2880 aaatttccag atctttacag tgaccttgat aagtgttctg accagggcat aaggaattat 2940 gctgtttcag tgacatctct gaatgaagta ttcttgaacc tagaaggaaa atcagcaatt 3000 gatgaaccag attttgacat tgggaaacaa gagaaaatac atgtgacaag aaatactgga 3060 gatgagtctg aaatggaaca ggttctttgt tctcttcctg aaacaagaaa ggctgtcagt 3120 agtgcagctc tctggagacg acaaatctat gcagtggcaa cacttcgctt cttaaagtta 3180 aggcgtgaaa ggagagctct tttgtgtttg ttactagtac ttggaattgc ttttatcccc 3240 atcattctag agaagataat gtataaagta actcgtgaaa ctcattgttg ggagttttca 3300 cccagtatgt atttcctttc tctggaacaa atcccgaaga cgcctcttac cagcctgtta 3360 atcgttaata atacaggatc aaatattgaa gacctcgtgc attcactgaa gtgtcaggat 3420 atagttttgg aaatagatga ctttagaaac agaaatggct cagatgatcc ctcctacaat 3480 ggagccatca tagtgtctgg tgaccagaag gattacagat tttctgttgc gtgtaatacc 3540 aagaaattga attgttttcc tgttcttatg ggaattgtta gcaatgccct tatgggaatt 3600 tttaacttca cggagcttat tcaaacggag agcacttcat tttctcgtga tgacatagtg 3660 ctggatcttg gttttataga tgggtccata tttttgttgt tgatcacaaa ctgcgtttct 3720 ccttttatcg gcatgagcag catcagcgat tataaaaaaa atgttcaatc ccagttatgg 3780 atttcaggcc tctggccttc agcatactgg tgtggacagg ctctggtgga cattccatta 3840 tacttcttga ttctcttttc aatacattta atttactact tcatatttct gggattccag 3900 ctttcatggg aactcatgtt tgttttggtg gtatgcataa ttggttgtgc agtttctctt 3960 atattcctca catatgtgct ttcattcatc tttcgcaagt ggagaaaaaa taatggcttt 4020 tggtcttttg gcttttttat tatcttaata tgtgtatcca caattatggt atcaactcaa 4080 tatgaaaaac tcaacttaat tttgtgcatg attttcatac cttccttcac tttgctgggg 4140 tatgtcatgt tattgatcca gctcgacttt atgagaaact tggacagtct ggacaataga 4200 ataaatgaag tcaataaaac cattctttta acaaccttaa taccatacct tcagagtgtt 4260 attttccttt ttgtcataag gtgtctggaa atgaagtatg gaaatgaaat aatgaataaa 4320 gacccagttt tcagaatctc tccacggagt agagaaactc atcccaatcc ggaagagccc 4380 gaagaagaag atgaagatgt tcaagctgaa agagtccaag cagcaaatgc actcactgct 4440 ccaaacttgg aggaggaacc agtcataact gcaagctgtt tacacaagga atattatgag 4500 acaaagaaaa gttgcttttc aacaagaaag aagaaaatag ccatcagaaa tgtttccttt 4560 tgtgttaaaa aaggtgaagt tttgggatta ctaggacaca atggagctgg taaaagtact 4620 tccattaaaa tgataactgg gtgcacaaag ccaactgcag gagtggtggt gttacaaggc 4680 agcagagcat cagtaaggca acagcatgac aacagcctca agttcttggg gtactgccct 4740 caggagaact cactgtggcc caagcttaca atgaaagagc acttggagtt gtatgcagct 4800 gtgaaaggac tgggcaaaga agatgctgct ctcagtattt cacgattggt ggaagctctt 4860 aagctccagg aacaacttaa ggctcctgtg aaaactctat cagagggaat aaagagaaag 4920 ctgtgctttg tgctgagcat cctggggaac ccatcagtgg tgcttctaga tgagccgttc 4980 accgggatgg accccgaggg gcagcagcaa atgtggcaga tacttcaggc taccgttaaa 5040 aacaaggaga ggggcaccct cttgaccacc cattacatgt cagaggctga ggctgtgtgt 5100 gaccgtatgg ccatgatggt gtcaggaacg ctaaggtgta ttggttccat tcaacatctg 5160 aaaaacaagt ttggtagaga ttatttacta gaaataaaaa tgaaagaacc tacccaggtg 5220 gaagctctcc acacagagat tttgaagctt ttcccacagg ctgcttggca ggaaagatat 5280 tcctctttaa tggcgtataa gttacctgtg gaggatgtcc accctctatc tcgggccttt 5340 ttcaagttag aggcgatgaa acagaccttc aacctggagg aatacagcct ctctcaggct 5400 accttggagc aggtattctt agaactctgt aaagagcagg agctgggaaa tgttgatgat 5460 aaaattgata caacagttga atggaaactt ctcccacagg aagaccctta aaatgaagaa 5520 cctcctaaca ttcaatttta ggtcctacta cattgttagt ttccataatt ctacaagaat 5580 gtttcctttt acttcagtta acaaaagaaa acatttaata aacattcaat aatgattaca 5640 gttttcattt ttaaaaattt aggatgaagg aaacaaggaa atatagggaa aagtagtaga 5700 caaaattaac aaaatcagac atgttattca tccccaacat gggtctattt tgtgcttaaa 5760 aataatttaa aaatcataca atattaggtt ggttttcggt tattatcaat aaagctaaca 5820 ctgagaacat tttacaaata aaaatatgag ttttttagcc tgaacttcaa atgtatcagc 5880 tatttttaaa cattatttac tcggattcta atttaatgtg acattgacta taagaaggtc 5940 tgataaactg atgaaatggc acagcataac atttaattat aatgacattc tgattataaa 6000 ataaatgcat gtgaatttta gtacatattg aagttatatg gaagaagata gccataatct 6060 gtaagaaagt accgcagtta atattttctt tagccaactt atattcaatg tattttttat 6120 ggatcctttt tcaaaggtag tatcagtagg catagtcatt ttctgtatct tttcacctca 6180 c 6181 5 1642 PRT Homo sapiens UNSURE 1147 Xaa=unknown, may be any amino acid 5 Met Ser Thr Ala Ile Arg Glu Val Gly Val Trp Arg Gln Thr Arg Thr 1 5 10 15 Leu Leu Leu Lys Asn Tyr Leu Ile Lys Cys Arg Thr Lys Lys Ser Ser 20 25 30 Val Gln Glu Ile Leu Phe Pro Leu Phe Phe Leu Phe Trp Leu Ile Leu 35 40 45 Ile Ser Met Met His Pro Asn Lys Lys Tyr Glu Glu Val Pro Asn Ile 50 55 60 Glu Leu Asn Pro Met Asp Lys Phe Thr Leu Ser Asn Leu Ile Leu Gly 65 70 75 80 Tyr Thr Pro Val Thr Asn Ile Thr Ser Ser Ile Met Gln Lys Val Ser 85 90 95 Thr Asp His Leu Pro Asp Val Ile Ile Thr Glu Glu Tyr Thr Asn Glu 100 105 110 Lys Glu Met Leu Thr Ser Ser Leu Ser Lys Pro Ser Asn Phe Val Gly 115 120 125 Val Val Phe Lys Asp Ser Met Ser Tyr Glu Leu Arg Phe Phe Pro Asp 130 135 140 Met Ile Pro Val Ser Ser Ile Tyr Met Asp Ser Arg Ala Gly Cys Ser 145 150 155 160 Lys Ser Cys Glu Ala Ala Gln Tyr Trp Ser Ser Gly Phe Thr Val Leu 165 170 175 Gln Ala Ser Ile Asp Ala Ala Ile Ile Gln Leu Lys Thr Asn Val Ser 180 185 190 Leu Trp Lys Glu Leu Glu Ser Thr Lys Ala Val Ile Met Gly Glu Thr 195 200 205 Ala Val Val Glu Ile Asp Thr Phe Pro Arg Gly Val Ile Leu Ile Tyr 210 215 220 Leu Val Ile Ala Phe Ser Pro Phe Gly Tyr Phe Leu Ala Ile His Ile 225 230 235 240 Val Ala Glu Lys Glu Lys Lys Ile Lys Glu Phe Leu Lys Ile Met Gly 245 250 255 Leu His Asp Thr Ala Phe Trp Leu Ser Trp Val Leu Leu Tyr Thr Ser 260 265 270 Leu Ile Phe Leu Met Ser Leu Leu Met Ala Val Ile Ala Thr Ala Ser 275 280 285 Leu Leu Phe Pro Gln Ser Ser Ser Ile Val Ile Phe Leu Leu Phe Phe 290 295 300 Leu Tyr Gly Leu Ser Ser Val Phe Phe Ala Leu Met Leu Thr Pro Leu 305 310 315 320 Phe Lys Lys Ser Lys His Val Gly Ile Val Glu Phe Phe Val Thr Val 325 330 335 Ala Phe Gly Phe Ile Gly Leu Met Ile Ile Leu Ile Glu Ser Phe Pro 340 345 350 Lys Ser Leu Val Trp Leu Phe Ser Pro Phe Cys His Cys Thr Phe Val 355 360 365 Ile Gly Ile Ala Gln Val Met His Leu Glu Asp Phe Asn Glu Gly Ala 370 375 380 Ser Phe Ser Asn Leu Thr Ala Gly Pro Tyr Pro Leu Ile Ile Thr Ile 385 390 395 400 Ile Met Leu Thr Leu Asn Ser Ile Phe Tyr Val Leu Leu Ala Val Tyr 405 410 415 Leu Asp Gln Val Ile Pro Gly Glu Phe Gly Leu Arg Arg Ser Ser Leu 420 425 430 Tyr Phe Leu Lys Pro Ser Tyr Trp Ser Lys Ser Lys Arg Asn Tyr Glu 435 440 445 Glu Leu Ser Glu Gly Asn Val Asn Gly Asn Ile Ser Phe Ser Glu Ile 450 455 460 Ile Glu Pro Val Ser Ser Glu Phe Val Gly Lys Glu Ala Ile Arg Ile 465 470 475 480 Ser Gly Ile Gln Lys Thr Tyr Arg Lys Lys Gly Glu Asn Val Glu Ala 485 490 495 Leu Arg Asn Leu Ser Phe Asp Ile Tyr Glu Gly Gln Ile Thr Ala Leu 500 505 510 Leu Gly His Ser Gly Thr Gly Lys Ser Thr Leu Met Asn Ile Leu Cys 515 520 525 Gly Leu Cys Pro Pro Ser Asp Gly Phe Ala Ser Ile Tyr Gly His Arg 530 535 540 Val Ser Glu Ile Asp Glu Met Phe Glu Ala Arg Lys Met Ile Gly Ile 545 550 555 560 Cys Pro Gln Leu Asp Ile His Phe Asp Val Leu Thr Val Glu Glu Asn 565 570 575 Leu Ser Ile Leu Ala Ser Ile Lys Gly Ile Pro Ala Asn Asn Ile Ile 580 585 590 Gln Glu Val Gln Lys Val Leu Leu Asp Leu Asp Met Gln Thr Ile Lys 595 600 605 Asp Asn Gln Ala Lys Lys Leu Ser Gly Gly Gln Lys Arg Lys Leu Ser 610 615 620 Leu Gly Ile Ala Val Leu Gly Asn Pro Lys Ile Leu Leu Leu Asp Glu 625 630 635 640 Pro Thr Ala Gly Met Asp Pro Cys Ser Arg His Ile Val Trp Asn Leu 645 650 655 Leu Lys Tyr Arg Lys Ala Asn Arg Val Thr Val Phe Ser Thr His Phe 660 665 670 Met Asp Glu Ala Asp Ile Leu Ala Asp Arg Lys Ala Val Ile Ser Gln 675 680 685 Gly Met Leu Lys Cys Val Gly Ser Ser Met Phe Leu Lys Ser Lys Trp 690 695 700 Gly Ile Gly Tyr Arg Leu Ser Met Tyr Ile Asp Lys Tyr Cys Ala Thr 705 710 715 720 Glu Ser Leu Ser Ser Leu Val Lys Gln His Ile Pro Gly Ala Thr Leu 725 730 735 Leu Gln Gln Asn Asp Gln Gln Leu Val Tyr Ser Leu Pro Phe Lys Asp 740 745 750 Met Asp Lys Phe Ser Gly Leu Phe Ser Ala Leu Asp Ser His Ser Asn 755 760 765 Leu Gly Val Ile Ser Tyr Gly Val Ser Met Thr Thr Leu Glu Asp Val 770 775 780 Phe Leu Lys Leu Glu Val Glu Ala Glu Ile Asp Gln Ala Asp Tyr Ser 785 790 795 800 Val Phe Thr Gln Gln Pro Leu Glu Glu Glu Met Asp Ser Lys Ser Phe 805 810 815 Asp Glu Met Glu Gln Ser Leu Leu Ile Leu Ser Glu Thr Lys Ala Ser 820 825 830 Leu Val Ser Thr Met Ser Leu Trp Lys Gln Gln Met Tyr Thr Ile Ala 835 840 845 Lys Phe His Phe Phe Thr Leu Lys Arg Glu Ser Lys Ser Val Arg Ser 850 855 860 Val Leu Leu Leu Leu Leu Ile Phe Phe Thr Val Gln Ile Phe Met Phe 865 870 875 880 Leu Val His His Ser Phe Lys Asn Ala Val Val Pro Ile Lys Leu Val 885 890 895 Pro Asp Leu Tyr Phe Leu Lys Pro Gly Asp Lys Pro His Lys Tyr Lys 900 905 910 Thr Ser Leu Leu Leu Gln Asn Ser Ala Asp Ser Asp Ile Ser Asp Leu 915 920 925 Ile Ser Phe Phe Thr Ser Gln Asn Ile Met Val Thr Met Ile Asn Asp 930 935 940 Ser Asp Tyr Val Ser Val Ala Pro His Ser Ala Ala Leu Asn Val Met 945 950 955 960 His Ser Glu Lys Asp Tyr Val Phe Ala Ala Val Phe Asn Ser Thr Met 965 970 975 Val Tyr Ser Leu Pro Ile Leu Val Asn Ile Ile Ser Asn Tyr Tyr Leu 980 985 990 Tyr His Leu Asn Val Thr Glu Thr Ile Gln Ile Trp Ser Thr Pro Phe 995 1000 1005 Phe Gln Glu Ile Thr Asp Ile Val Phe Lys Ile Glu Leu Tyr Phe Gln 1010 1015 1020 Ala Ala Leu Leu Gly Ile Ile Val Thr Ala Met Pro Pro Tyr Phe Ala 1025 1030 1035 1040 Met Glu Asn Ala Glu Asn His Lys Ile Lys Ala Tyr Thr Gln Leu Lys 1045 1050 1055 Leu Ser Gly Leu Leu Pro Ser Ala Tyr Trp Ile Gly Gln Ala Val Val 1060 1065 1070 Asp Ile Pro Leu Phe Phe Ile Ile Leu Ile Leu Met Leu Gly Ser Leu 1075 1080 1085 Leu Ala Phe His Tyr Gly Leu Tyr Phe Tyr Thr Val Lys Phe Leu Ala 1090 1095 1100 Val Val Phe Cys Leu Ile Gly Tyr Val Pro Ser Val Ile Leu Phe Thr 1105 1110 1115 1120 Tyr Ile Ala Ser Phe Thr Phe Lys Lys Ile Leu Asn Thr Lys Glu Phe 1125 1130 1135 Trp Ser Phe Ile Tyr Ser Val Ala Ala Leu Xaa Cys Ile Ala Ile Thr 1140 1145 1150 Glu Ile Thr Phe Phe Met Gly Tyr Thr Ile Ala Thr Ile Leu His Tyr 1155 1160 1165 Ala Phe Cys Ile Ile Ile Pro Ile Tyr Pro Leu Leu Gly Cys Leu Ile 1170 1175 1180 Ser Phe Ile Lys Ile Ser Trp Lys Asn Val Arg Lys Asn Val Asp Thr 1185 1190 1195 1200 Tyr Asn Pro Trp Asp Arg Leu Ser Val Ala Val Ile Ser Pro Tyr Leu 1205 1210 1215 Gln Cys Val Leu Trp Ile Phe Leu Leu Gln Tyr Tyr Glu Lys Lys Tyr 1220 1225 1230 Gly Gly Arg Ser Ile Arg Lys Asp Pro Phe Phe Arg Asn Leu Ser Thr 1235 1240 1245 Lys Ser Lys Asn Arg Lys Leu Pro Glu Pro Pro Asp Asn Glu Asp Glu 1250 1255 1260 Asp Glu Asp Val Lys Ala Glu Arg Leu Lys Val Lys Glu Leu Met Gly 1265 1270 1275 1280 Cys Gln Cys Cys Glu Glu Lys Pro Ser Ile Met Val Ser Asn Leu His 1285 1290 1295 Lys Glu Tyr Asp Asp Lys Lys Asp Phe Leu Leu Ser Arg Lys Val Lys 1300 1305 1310 Lys Val Ala Thr Lys Tyr Ile Ser Phe Cys Val Lys Lys Gly Glu Ile 1315 1320 1325 Leu Gly Leu Leu Gly Pro Asn Gly Ala Gly Lys Ser Thr Ile Ile Asn 1330 1335 1340 Ile Leu Val Gly Asp Ile Glu Pro Thr Ser Gly Gln Val Phe Leu Gly 1345 1350 1355 1360 Asp Tyr Ser Ser Glu Thr Ser Glu Asp Asp Asp Ser Leu Lys Cys Met 1365 1370 1375 Gly Tyr Cys Pro Gln Ile Asn Pro Leu Trp Pro Asp Thr Thr Leu Gln 1380 1385 1390 Glu His Phe Glu Ile Tyr Gly Ala Val Lys Gly Met Ser Ala Ser Asp 1395 1400 1405 Met Lys Glu Val Ile Ser Arg Ile Thr His Ala Leu Asp Leu Lys Glu 1410 1415 1420 His Leu Gln Lys Thr Val Lys Lys Leu Pro Ala Gly Ile Lys Arg Lys 1425 1430 1435 1440 Leu Cys Phe Ala Leu Ser Met Leu Gly Asn Pro Gln Ile Thr Leu Leu 1445 1450 1455 Asp Glu Pro Ser Thr Gly Met Asp Pro Lys Ala Lys Gln His Met Trp 1460 1465 1470 Arg Ala Ile Arg Thr Ala Phe Lys Asn Arg Lys Arg Ala Ala Ile Leu 1475 1480 1485 Thr Thr His Tyr Met Glu Glu Ala Glu Ala Val Cys Asp Arg Val Ala 1490 1495 1500 Ile Met Val Ser Gly Gln Leu Arg Cys Ile Gly Thr Val Gln His Leu 1505 1510 1515 1520 Lys Ser Lys Phe Gly Lys Gly Tyr Phe Leu Glu Ile Lys Leu Lys Asp 1525 1530 1535 Trp Ile Glu Asn Leu Glu Val Asp Arg Leu Gln Arg Glu Ile Gln Tyr 1540 1545 1550 Ile Phe Pro Asn Ala Ser Arg Gln Glu Ser Phe Ser Ser Ile Leu Ala 1555 1560 1565 Tyr Lys Ile Pro Lys Glu Asp Val Gln Ser Leu Ser Gln Ser Phe Phe 1570 1575 1580 Lys Leu Glu Glu Ala Lys His Ala Phe Ala Ile Glu Glu Tyr Ser Phe 1585 1590 1595 1600 Ser Gln Ala Thr Leu Glu Gln Val Phe Val Glu Leu Thr Lys Glu Gln 1605 1610 1615 Glu Glu Glu Asp Asn Ser Cys Gly Thr Leu Asn Ser Thr Leu Trp Trp 1620 1625 1630 Glu Arg Thr Gln Glu Asp Arg Val Val Phe 1635 1640 6 1617 PRT Homo sapiens 6 Met Asn Met Lys Gln Lys Ser Val Tyr Gln Gln Thr Lys Ala Leu Leu 1 5 10 15 Cys Lys Asn Phe Leu Lys Lys Trp Arg Met Lys Arg Glu Ser Leu Leu 20 25 30 Glu Trp Gly Leu Ser Ile Leu Leu Gly Leu Cys Ile Ala Leu Phe Ser 35 40 45 Ser Ser Met Arg Asn Val Gln Phe Pro Gly Met Ala Pro Gln Asn Leu 50 55 60 Gly Arg Val Asp Lys Phe Asn Ser Ser Ser Leu Met Val Val Tyr Thr 65 70 75 80 Pro Ile Ser Asn Leu Thr Gln Gln Ile Met Asn Lys Thr Ala Leu Ala 85 90 95 Pro Leu Leu Lys Gly Thr Ser Val Ile Gly Ala Pro Asn Lys Thr His 100 105 110 Met Asp Glu Ile Leu Leu Glu Asn Leu Pro Tyr Ala Met Gly Ile Ile 115 120 125 Phe Asn Glu Thr Phe Ser Tyr Lys Leu Ile Phe Phe Gln Gly Tyr Asn 130 135 140 Ser Pro Leu Trp Lys Glu Asp Phe Ser Ala His Cys Trp Asp Gly Tyr 145 150 155 160 Gly Glu Phe Ser Cys Thr Leu Thr Lys Tyr Trp Asn Arg Gly Phe Val 165 170 175 Ala Leu Gln Thr Ala Ile Asn Thr Ala Ile Ile Glu Ile Thr Thr Asn 180 185 190 His Pro Val Met Glu Glu Leu Met Ser Val Thr Ala Ile Thr Met Lys 195 200 205 Thr Leu Pro Phe Ile Thr Lys Asn Leu Leu His Asn Glu Met Phe Ile 210 215 220 Leu Phe Phe Leu Leu His Phe Ser Pro Leu Val Tyr Phe Ile Ser Leu 225 230 235 240 Asn Val Thr Lys Glu Arg Lys Lys Ser Lys Asn Leu Met Lys Met Met 245 250 255 Gly Leu Gln Asp Ser Ala Phe Trp Leu Ser Trp Gly Leu Ile Tyr Ala 260 265 270 Gly Phe Ile Phe Ile Ile Ser Ile Phe Ile Thr Ile Ile Ile Thr Phe 275 280 285 Thr Gln Ile Ile Val Met Thr Gly Phe Met Val Ile Phe Ile Leu Phe 290 295 300 Phe Leu Tyr Gly Leu Ser Leu Val Ala Leu Val Phe Leu Met Ser Val 305 310 315 320 Leu Leu Lys Lys Ala Val Leu Thr Asn Leu Val Val Phe Leu Leu Thr 325 330 335 Leu Phe Trp Gly Cys Leu Gly Phe Thr Val Phe Tyr Glu Gln Leu Pro 340 345 350 Ser Ser Leu Glu Trp Ile Leu Asn Ile Cys Ser Pro Phe Ala Phe Thr 355 360 365 Thr Gly Met Ile Gln Ile Ile Lys Leu Asp Tyr Asn Leu Asn Gly Val 370 375 380 Ile Phe Pro Asp Pro Ser Gly Asp Ser Tyr Thr Met Ile Ala Thr Phe 385 390 395 400 Ser Met Leu Leu Leu Asp Gly Leu Ile Tyr Leu Leu Leu Ala Leu Tyr 405 410 415 Phe Asp Lys Ile Leu Pro Tyr Gly Asp Glu Arg His Tyr Ser Pro Leu 420 425 430 Phe Phe Leu Asn Ser Ser Ser Cys Phe Gln His Gln Arg Thr Asn Ala 435 440 445 Lys Val Ile Glu Lys Glu Ile Asp Ala Glu His Pro Ser Asp Asp Tyr 450 455 460 Phe Glu Pro Val Ala Pro Glu Phe Gln Gly Lys Glu Ala Ile Arg Ile 465 470 475 480 Arg Asn Val Lys Lys Glu Tyr Lys Gly Lys Ser Gly Lys Val Glu Ala 485 490 495 Leu Lys Gly Leu Leu Phe Asp Ile Tyr Glu Gly Gln Ile Thr Ala Ile 500 505 510 Leu Gly His Ser Gly Ala Gly Lys Ser Ser Leu Leu Asn Ile Leu Asn 515 520 525 Gly Leu Ser Val Pro Thr Glu Gly Ser Val Thr Ile Tyr Asn Lys Asn 530 535 540 Leu Ser Glu Met Gln Asp Leu Glu Glu Ile Arg Lys Ile Thr Gly Val 545 550 555 560 Cys Pro Gln Phe Asn Val Gln Phe Asp Ile Leu Thr Val Lys Glu Asn 565 570 575 Leu Ser Leu Phe Ala Lys Ile Lys Gly Ile His Leu Lys Glu Val Glu 580 585 590 Gln Glu Val Gln Arg Ile Leu Leu Glu Leu Asp Met Gln Asn Ile Gln 595 600 605 Asp Asn Leu Ala Lys His Leu Ser Glu Gly Gln Lys Arg Lys Leu Thr 610 615 620 Phe Gly Ile Thr Ile Leu Gly Asp Pro Gln Ile Leu Leu Leu Asp Glu 625 630 635 640 Pro Thr Thr Gly Leu Asp Pro Phe Ser Arg Asp Gln Val Trp Ser Leu 645 650 655 Leu Arg Glu Arg Arg Ala Asp His Val Ile Leu Phe Ser Thr Gln Ser 660 665 670 Met Asp Glu Ala Asp Ile Leu Ala Asp Arg Lys Val Ile Met Ser Asn 675 680 685 Gly Arg Leu Lys Cys Ala Gly Ser Ser Met Phe Leu Lys Arg Arg Trp 690 695 700 Gly Leu Gly Tyr His Leu Ser Leu His Arg Asn Glu Ile Cys Asn Pro 705 710 715 720 Glu Gln Ile Thr Ser Phe Ile Thr His His Ile Pro Asp Ala Lys Leu 725 730 735 Lys Thr Glu Asn Lys Glu Lys Leu Val Tyr Thr Leu Pro Leu Glu Arg 740 745 750 Thr Asn Thr Phe Pro Asp Leu Phe Ser Asp Leu Asp Lys Cys Ser Asp 755 760 765 Gln Gly Val Thr Gly Tyr Asp Ile Ser Met Ser Thr Leu Asn Glu Val 770 775 780 Phe Met Lys Leu Glu Gly Gln Ser Thr Ile Glu Gln Asp Phe Glu Gln 785 790 795 800 Val Glu Met Ile Arg Asp Ser Glu Ser Leu Asn Glu Met Glu Leu Ala 805 810 815 His Ser Ser Phe Ser Glu Met Gln Thr Ala Val Ser Asp Met Gly Leu 820 825 830 Trp Arg Met Gln Val Phe Ala Met Ala Arg Leu Arg Phe Leu Lys Leu 835 840 845 Lys Arg Gln Thr Lys Val Leu Leu Thr Leu Leu Leu Val Phe Gly Ile 850 855 860 Ala Ile Phe Pro Leu Ile Val Glu Asn Ile Ile Tyr Ala Met Leu Asn 865 870 875 880 Glu Lys Ile Asp Trp Glu Phe Lys Asn Glu Leu Tyr Phe Leu Ser Pro 885 890 895 Gly Gln Leu Pro Gln Glu Pro Arg Thr Ser Leu Leu Ile Ile Asn Asn 900 905 910 Thr Glu Ser Asn Ile Glu Asp Phe Ile Lys Ser Leu Lys His Gln Asn 915 920 925 Ile Leu Leu Glu Val Asp Asp Phe Glu Asn Arg Asn Gly Thr Asp Gly 930 935 940 Leu Ser Tyr Asn Gly Ala Ile Ile Val Ser Gly Lys Gln Lys Asp Tyr 945 950 955 960 Arg Phe Ser Val Val Cys Asn Thr Lys Arg Leu His Cys Phe Pro Ile 965 970 975 Leu Met Asn Ile Ile Ser Asn Gly Leu Leu Gln Met Phe Asn His Thr 980 985 990 Gln His Ile Arg Ile Glu Ser Ser Pro Phe Pro Leu Ser His Ile Gly 995 1000 1005 Leu Trp Thr Gly Leu Pro Asp Gly Ser Phe Phe Leu Phe Leu Val Leu 1010 1015 1020 Cys Ser Ile Ser Pro Tyr Ile Thr Met Gly Ser Ile Ser Asp Tyr Lys 1025 1030 1035 1040 Lys Asn Ala Lys Ser Gln Leu Trp Ile Ser Gly Leu Tyr Thr Ser Ala 1045 1050 1055 Tyr Trp Cys Gly Gln Ala Leu Val Asp Val Ser Phe Phe Ile Leu Ile 1060 1065 1070 Leu Leu Leu Met Tyr Leu Ile Phe Tyr Ile Glu Asn Met Gln Tyr Leu 1075 1080 1085 Leu Ile Thr Ser Gln Ile Val Phe Ala Leu Val Ile Val Thr Pro Gly 1090 1095 1100 Tyr Ala Ala Ser Leu Val Phe Phe Ile Tyr Met Ile Ser Phe Ile Phe 1105 1110 1115 1120 Arg Lys Arg Arg Lys Asn Ser Gly Leu Trp Ser Phe Tyr Phe Phe Phe 1125 1130 1135 Ala Ser Thr Ile Met Phe Ser Ile Thr Leu Ile Asn His Phe Asp Leu 1140 1145 1150 Ser Ile Leu Ile Thr Thr Met Val Leu Val Pro Ser Tyr Thr Leu Leu 1155 1160 1165 Gly Phe Lys Thr Phe Leu Glu Val Arg Asp Gln Glu His Tyr Arg Glu 1170 1175 1180 Phe Pro Glu Ala Asn Phe Glu Leu Ser Ala Thr Asp Phe Leu Val Cys 1185 1190 1195 1200 Phe Ile Pro Tyr Phe Gln Thr Leu Leu Phe Val Phe Val Leu Arg Cys 1205 1210 1215 Met Glu Leu Lys Cys Gly Lys Lys Arg Met Arg Lys Asp Pro Val Phe 1220 1225 1230 Arg Ile Ser Pro Gln Ser Arg Asp Ala Lys Pro Asn Pro Glu Glu Pro 1235 1240 1245 Ile Asp Glu Asp Glu Asp Ile Gln Thr Glu Arg Ile Arg Thr Ala Thr 1250 1255 1260 Ala Leu Thr Thr Ser Ile Leu Asp Glu Lys Pro Val Ile Ile Ala Ser 1265 1270 1275 1280 Cys Leu His Lys Glu Tyr Ala Gly Gln Lys Lys Ser Cys Phe Ser Lys 1285 1290 1295 Arg Lys Lys Lys Ile Ala Ala Arg Asn Ile Ser Phe Cys Val Gln Glu 1300 1305 1310 Gly Glu Ile Leu Gly Leu Leu Gly Pro Ser Gly Ala Gly Lys Ser Ser 1315 1320 1325 Ser Ile Arg Met Ile Ser Gly Ile Thr Lys Pro Thr Ala Gly Glu Val 1330 1335 1340 Glu Leu Lys Gly Cys Ser Ser Val Leu Gly His Leu Gly Tyr Cys Pro 1345 1350 1355 1360 Gln Glu Asn Val Leu Trp Pro Met Leu Thr Leu Arg Glu His Leu Glu 1365 1370 1375 Val Tyr Ala Ala Val Lys Gly Leu Arg Lys Ala Asp Ala Arg Leu Ala 1380 1385 1390 Ile Ala Arg Leu Val Ser Ala Phe Lys Leu His Glu Gln Leu Asn Val 1395 1400 1405 Pro Val Gln Lys Leu Thr Ala Gly Ile Thr Arg Lys Leu Cys Phe Val 1410 1415 1420 Leu Ser Leu Leu Gly Asn Ser Pro Val Leu Leu Leu Asp Glu Pro Ser 1425 1430 1435 1440 Thr Gly Ile Asp Pro Thr Gly Gln Gln Gln Met Trp Gln Ala Ile Gln 1445 1450 1455 Ala Val Val Lys Asn Thr Glu Arg Gly Val Leu Leu Thr Thr His Asn 1460 1465 1470 Leu Ala Glu Ala Glu Ala Leu Cys Asp Arg Val Ala Ile Met Val Ser 1475 1480 1485 Gly Arg Leu Arg Cys Ile Gly Ser Ile Gln His Leu Lys Asn Lys Leu 1490 1495 1500 Gly Lys Asp Tyr Ile Leu Glu Leu Lys Val Lys Glu Thr Ser Gln Val 1505 1510 1515 1520 Thr Leu Val His Thr Glu Ile Leu Lys Leu Phe Pro Gln Ala Ala Gly 1525 1530 1535 Gln Glu Arg Tyr Ser Ser Leu Leu Thr Tyr Lys Leu Pro Val Ala Asp 1540 1545 1550 Val Tyr Pro Leu Ser Gln Thr Phe His Lys Leu Glu Ala Val Lys His 1555 1560 1565 Asn Phe Asn Leu Glu Glu Tyr Ser Leu Ser Gln Cys Thr Leu Glu Lys 1570 1575 1580 Val Phe Leu Glu Leu Ser Lys Glu Gln Glu Val Gly Asn Phe Asp Glu 1585 1590 1595 1600 Glu Ile Asp Thr Thr Met Arg Trp Lys Leu Leu Pro His Ser Asp Glu 1605 1610 1615 Pro 7 1624 PRT Homo sapiens 7 Met Ser Lys Arg Arg Met Ser Val Gly Gln Gln Thr Trp Ala Leu Leu 1 5 10 15 Cys Lys Asn Cys Leu Lys Lys Trp Arg Met Lys Arg Gln Thr Leu Leu 20 25 30 Glu Trp Leu Phe Ser Phe Leu Leu Val Leu Phe Leu Tyr Leu Phe Phe 35 40 45 Ser Asn Leu His Gln Val His Asp Thr Pro Gln Met Ser Ser Met Asp 50 55 60 Leu Gly Arg Val Asp Ser Phe Asn Asp Thr Asn Tyr Val Ile Ala Phe 65 70 75 80 Ala Pro Glu Ser Lys Thr Thr Gln Glu Ile Met Asn Lys Val Ala Ser 85 90 95 Ala Pro Phe Leu Lys Gly Arg Thr Ile Met Gly Trp Pro Asp Glu Lys 100 105 110 Ser Met Asp Glu Leu Asp Leu Asn Tyr Ser Ile Asp Ala Val Arg Val 115 120 125 Ile Phe Thr Asp Thr Phe Ser Tyr His Leu Lys Phe Ser Trp Gly His 130 135 140 Arg Ile Pro Met Met Lys Glu His Arg Asp His Ser Ala His Cys Gln 145 150 155 160 Ala Val Asn Glu Lys Met Lys Cys Glu Gly Ser Glu Phe Trp Glu Lys 165 170 175 Gly Phe Val Ala Phe Gln Ala Ala Ile Asn Ala Ala Ile Ile Glu Ile 180 185 190 Ala Thr Asn His Ser Val Met Glu Gln Leu Met Ser Val Thr Gly Val 195 200 205 His Met Lys Ile Leu Pro Phe Val Ala Gln Gly Gly Val Ala Thr Asp 210 215 220 Phe Phe Ile Phe Phe Cys Ile Ile Ser Phe Ser Thr Phe Ile Tyr Tyr 225 230 235 240 Val Ser Val Asn Val Thr Gln Glu Arg Gln Tyr Ile Thr Ser Leu Met 245 250 255 Thr Met Met Gly Leu Arg Glu Ser Ala Phe Trp Leu Ser Trp Gly Leu 260 265 270 Met Tyr Ala Gly Phe Ile Leu Ile Met Ala Thr Leu Met Ala Leu Ile 275 280 285 Val Lys Ser Ala Gln Ile Val Val Leu Thr Gly Phe Val Met Val Phe 290 295 300 Thr Leu Phe Leu Leu Tyr Gly Leu Ser Leu Ile Thr Leu Ala Phe Leu 305 310 315 320 Met Ser Val Leu Ile Lys Lys Pro Phe Leu Thr Gly Leu Val Val Phe 325 330 335 Leu Leu Ile Val Phe Trp Gly Ile Leu Gly Phe Pro Ala Leu Tyr Thr 340 345 350 His Leu Pro Ala Phe Leu Glu Trp Thr Leu Cys Leu Leu Ser Pro Phe 355 360 365 Ala Phe Thr Val Gly Met Ala Gln Leu Ile His Leu Asp Tyr Asp Val 370 375 380 Asn Ser Asn Ala His Leu Asp Ser Ser Gln Asn Pro Tyr Leu Ile Ile 385 390 395 400 Ala Thr Leu Phe Met Leu Val Phe Asp Thr Leu Leu Tyr Leu Val Leu 405 410 415 Thr Leu Tyr Phe Asp Lys Ile Leu Pro Ala Glu Tyr Gly His Arg Cys 420 425 430 Ser Pro Leu Phe Phe Leu Lys Ser Cys Phe Trp Phe Gln His Gly Arg 435 440 445 Ala Asn His Val Val Leu Glu Asn Glu Thr Asp Ser Asp Pro Thr Pro 450 455 460 Asn Asp Cys Phe Glu Pro Val Ser Pro Glu Phe Cys Gly Lys Glu Ala 465 470 475 480 Ile Arg Ile Lys Asn Leu Lys Lys Glu Tyr Ala Gly Lys Cys Glu Arg 485 490 495 Val Glu Ala Leu Lys Gly Val Val Phe Asp Ile Tyr Glu Gly Gln Ile 500 505 510 Thr Ala Leu Leu Gly His Ser Gly Ala Gly Lys Thr Thr Leu Leu Asn 515 520 525 Ile Leu Ser Gly Leu Ser Val Pro Thr Ser Gly Ser Val Thr Val Tyr 530 535 540 Asn His Thr Leu Ser Arg Met Ala Asp Ile Glu Asn Ile Ser Lys Phe 545 550 555 560 Thr Gly Phe Cys Pro Gln Ser Asn Val Gln Phe Gly Phe Leu Thr Val 565 570 575 Lys Glu Asn Leu Arg Leu Phe Ala Lys Ile Lys Gly Ile Leu Pro His 580 585 590 Glu Val Glu Lys Glu Val Gln Arg Val Val Gln Glu Leu Glu Met Glu 595 600 605 Asn Ile Gln Asp Ile Leu Ala Gln Asn Leu Ser Gly Gly Gln Asn Arg 610 615 620 Lys Leu Thr Phe Gly Ile Ala Ile Leu Gly Asp Pro Gln Val Leu Leu 625 630 635 640 Leu Asp Glu Pro Thr Ala Gly Leu Asp Pro Leu Ser Arg His Arg Ile 645 650 655 Trp Asn Leu Leu Lys Glu Gly Lys Ser Asp Arg Val Ile Leu Phe Ser 660 665 670 Thr Gln Phe Ile Asp Glu Ala Asp Ile Leu Ala Asp Arg Lys Val Phe 675 680 685 Ile Ser Asn Gly Lys Leu Lys Cys Ala Gly Ser Ser Leu Phe Leu Lys 690 695 700 Lys Lys Trp Gly Ile Gly Tyr His Leu Ser Leu His Leu Asn Glu Arg 705 710 715 720 Cys Asp Pro Glu Ser Ile Thr Ser Leu Val Lys Gln His Ile Ser Asp 725 730 735 Ala Lys Leu Thr Ala Gln Ser Glu Glu Lys Leu Val Tyr Ile Leu Pro 740 745 750 Leu Glu Arg Thr Asn Lys Phe Pro Glu Leu Tyr Arg Asp Leu Asp Arg 755 760 765 Cys Ser Asn Gln Gly Ile Glu Asp Tyr Gly Val Ser Ile Thr Thr Leu 770 775 780 Asn Glu Val Phe Leu Lys Leu Glu Gly Lys Ser Thr Ile Asp Glu Ser 785 790 795 800 Asp Ile Gly Ile Trp Gly Gln Leu Gln Thr Asp Gly Ala Lys Asp Ile 805 810 815 Gly Ser Leu Val Glu Leu Glu Gln Val Leu Ser Ser Phe His Glu Thr 820 825 830 Arg Lys Thr Ile Ser Gly Val Ala Leu Trp Arg Gln Gln Val Cys Ala 835 840 845 Ile Ala Lys Val Arg Phe Leu Lys Leu Lys Lys Glu Arg Lys Ser Leu 850 855 860 Trp Thr Ile Leu Leu Leu Phe Gly Ile Ser Phe Ile Pro Gln Leu Leu 865 870 875 880 Glu His Leu Phe Tyr Glu Ser Tyr Gln Lys Ser Tyr Pro Trp Glu Leu 885 890 895 Ser Pro Asn Thr Tyr Phe Leu Ser Pro Gly Gln Gln Pro Gln Asp Pro 900 905 910 Leu Thr His Leu Leu Val Ile Asn Lys Thr Gly Ser Thr Ile Asp Asn 915 920 925 Phe Leu His Ser Leu Arg Arg Gln Asn Ile Ala Ile Glu Val Asp Ala 930 935 940 Phe Gly Thr Arg Asn Gly Thr Asp Asp Pro Ser Tyr Asn Gly Ala Ile 945 950 955 960 Ile Val Ser Gly Asp Glu Lys Asp His Arg Phe Ser Ile Ala Cys Asn 965 970 975 Thr Lys Arg Leu Asn Cys Phe Pro Val Leu Leu Asp Val Ile Ser Asn 980 985 990 Gly Leu Leu Gly Ile Phe Asn Ser Ser Glu His Ile Gln Thr Asp Arg 995 1000 1005 Ser Thr Phe Phe Glu Glu His Met Asp Tyr Glu Tyr Gly Tyr Arg Ser 1010 1015 1020 Asn Thr Phe Phe Trp Ile Pro Met Ala Ala Ser Phe Thr Pro Tyr Ile 1025 1030 1035 1040 Ala Met Ser Ser Ile Gly Asp Tyr Lys Lys Lys Ala His Ser Gln Leu 1045 1050 1055 Arg Ile Ser Gly Leu Tyr Pro Ser Ala Tyr Trp Phe Gly Gln Ala Leu 1060 1065 1070 Val Asp Val Ser Leu Tyr Phe Leu Ile Leu Leu Leu Met Gln Ile Met 1075 1080 1085 Asp Tyr Ile Phe Ser Pro Glu Glu Ile Ile Phe Ile Ile Gln Asn Leu 1090 1095 1100 Leu Ile Gln Ile Leu Cys Ser Ile Gly Tyr Val Ser Ser Leu Val Phe 1105 1110 1115 1120 Leu Thr Tyr Val Ile Ser Phe Ile Phe Arg Asn Gly Arg Lys Asn Ser 1125 1130 1135 Gly Ile Trp Ser Phe Phe Phe Leu Ile Val Val Ile Phe Ser Ile Val 1140 1145 1150 Ala Thr Asp Leu Asn Glu Tyr Gly Phe Leu Gly Leu Phe Phe Gly Thr 1155 1160 1165 Met Leu Ile Pro Pro Phe Thr Leu Ile Gly Ser Leu Phe Ile Phe Ser 1170 1175 1180 Glu Ile Ser Pro Asp Ser Met Asp Tyr Leu Gly Ala Ser Glu Ser Glu 1185 1190 1195 1200 Ile Val Tyr Leu Ala Leu Leu Ile Pro Tyr Leu His Phe Leu Ile Phe 1205 1210 1215 Leu Phe Ile Leu Arg Cys Leu Glu Met Asn Cys Arg Lys Lys Leu Met 1220 1225 1230 Arg Lys Asp Pro Val Phe Arg Ile Ser Pro Arg Ser Asn Ala Ile Phe 1235 1240 1245 Pro Asn Pro Glu Glu Pro Glu Gly Glu Glu Glu Asp Ile Gln Met Glu 1250 1255 1260 Arg Met Arg Thr Val Asn Ala Met Ala Val Arg Asp Phe Asp Glu Thr 1265 1270 1275 1280 Pro Val Ile Ile Ala Ser Cys Leu Arg Lys Glu Tyr Ala Gly Lys Lys 1285 1290 1295 Lys Asn Cys Phe Ser Lys Arg Lys Lys Thr Ile Ala Thr Arg Asn Val 1300 1305 1310 Ser Phe Cys Val Lys Lys Gly Glu Val Ile Gly Leu Leu Gly His Asn 1315 1320 1325 Gly Ala Gly Lys Ser Thr Thr Ile Lys Met Ile Thr Gly Asp Thr Lys 1330 1335 1340 Pro Thr Ala Gly Gln Val Ile Leu Lys Gly Ser Gly Gly Gly Glu Pro 1345 1350 1355 1360 Leu Gly Phe Leu Gly Tyr Cys Pro Gln Glu Asn Ala Leu Trp Pro Asn 1365 1370 1375 Leu Thr Val Arg Gln His Leu Glu Val Tyr Ala Ala Val Lys Gly Leu 1380 1385 1390 Arg Lys Gly Asp Ala Met Ile Ala Ile Thr Arg Leu Val Asp Ala Leu 1395 1400 1405 Lys Leu Gln Asp Gln Leu Lys Ala Pro Val Lys Thr Leu Ser Glu Gly 1410 1415 1420 Ile Lys Arg Lys Leu Arg Phe Val Leu Ser Ile Leu Gly Asn Pro Ser 1425 1430 1435 1440 Val Val Leu Leu Asp Glu Pro Ser Thr Gly Met Asp Pro Glu Gly Gln 1445 1450 1455 Gln Gln Met Trp Gln Val Ile Arg Ala Thr Phe Arg Asn Thr Glu Arg 1460 1465 1470 Gly Ala Leu Leu Thr Thr His Tyr Met Ala Glu Ala Glu Ala Val Cys 1475 1480 1485 Asp Arg Val Ala Ile Met Val Ser Gly Arg Leu Arg Cys Ile Gly Ser 1490 1495 1500 Ile Gln His Leu Lys Ser Lys Phe Gly Lys Asp Tyr Leu Leu Glu Met 1505 1510 1515 1520 Lys Leu Lys Asn Leu Ala Gln Met Glu Pro Leu His Ala Glu Ile Leu 1525 1530 1535 Arg Leu Phe Pro Gln Ala Ala Gln Gln Glu Arg Phe Ser Ser Leu Met 1540 1545 1550 Val Tyr Lys Leu Pro Val Glu Asp Val Arg Pro Leu Ser Gln Ala Phe 1555 1560 1565 Phe Lys Leu Glu Ile Val Lys Gln Ser Phe Asp Leu Glu Glu Tyr Ser 1570 1575 1580 Leu Ser Gln Ser Thr Leu Glu Gln Val Phe Leu Glu Leu Ser Lys Glu 1585 1590 1595 1600 Gln Glu Leu Gly Asp Leu Glu Glu Asp Phe Asp Pro Ser Val Lys Trp 1605 1610 1615 Lys Leu Leu Leu Gln Glu Glu Pro 1620 8 1543 PRT Homo sapiens UNSURE 181 Xaa=unknown, may be any amino acid 8 Met Asn Lys Met Ala Leu Ala Ser Phe Met Lys Gly Arg Thr Val Ile 1 5 10 15 Gly Thr Pro Asp Glu Glu Thr Met Asp Ile Glu Leu Pro Lys Lys Tyr 20 25 30 His Glu Met Val Gly Val Ile Phe Ser Asp Thr Phe Ser Tyr Arg Leu 35 40 45 Lys Phe Asn Trp Gly Tyr Arg Ile Pro Val Ile Lys Glu His Ser Glu 50 55 60 Tyr Thr Glu His Cys Trp Ala Met His Gly Glu Ile Phe Cys Tyr Leu 65 70 75 80 Ala Lys Tyr Trp Leu Lys Gly Phe Val Ala Phe Gln Ala Ala Ile Asn 85 90 95 Ala Ala Ile Ile Glu Val Thr Thr Asn His Ser Val Met Glu Glu Leu 100 105 110 Thr Ser Val Ile Gly Ile Asn Met Lys Ile Pro Pro Phe Ile Ser Lys 115 120 125 Gly Glu Ile Met Asn Glu Trp Phe His Phe Thr Cys Leu Val Ser Phe 130 135 140 Ser Ser Phe Ile Tyr Phe Ala Ser Leu Asn Val Ala Arg Glu Arg Gly 145 150 155 160 Lys Phe Lys Lys Leu Met Thr Val Met Gly Leu Arg Glu Ser Ala Phe 165 170 175 Trp Leu Ser Trp Xaa Leu Thr Tyr Ile Cys Phe Ile Phe Ile Met Ser 180 185 190 Ile Phe Met Ala Leu Val Ile Thr Ser Ile Ser Ile Val Phe His Thr 195 200 205 Gly Phe Met Val Ile Phe Thr Leu Tyr Ser Leu Tyr Gly Leu Ser Leu 210 215 220 Ile Ala Leu Ala Phe Leu Met Ser Val Leu Ile Arg Lys Pro Met Leu 225 230 235 240 Ala Gly Leu Ala Gly Phe Leu Phe Thr Val Phe Trp Gly Cys Leu Gly 245 250 255 Phe Thr Val Leu Tyr Arg Gln Leu Pro Leu Ser Leu Gly Trp Val Leu 260 265 270 Ser Leu Leu Ser Pro Phe Ala Phe Thr Ala Gly Met Ala Gln Val Thr 275 280 285 His Leu Asp Asn Tyr Leu Ser Gly Val Ile Phe Pro Asp Pro Ser Gly 290 295 300 Asp Ser Tyr Lys Met Ile Ala Thr Phe Phe Ile Leu Ala Phe Asp Thr 305 310 315 320 Leu Phe Tyr Leu Ile Phe Thr Leu Tyr Phe Glu Arg Val Leu Pro Asp 325 330 335 Lys Asp Gly His Gly Asp Ser Pro Leu Phe Phe Leu Lys Ser Ser Phe 340 345 350 Trp Ser Lys His Gln Asn Thr His His Glu Ile Phe Glu Asn Glu Ile 355 360 365 Asn Pro Glu His Ser Ser Asp Asp Ser Phe Glu Pro Val Ser Pro Glu 370 375 380 Phe His Gly Lys Glu Ala Ile Arg Ile Arg Asn Val Ile Lys Glu Tyr 385 390 395 400 Asn Gly Lys Thr Gly Lys Val Glu Ala Leu Gln Gly Ile Phe Phe Asp 405 410 415 Ile Tyr Glu Gly Gln Ile Thr Ala Ile Leu Gly His Asn Gly Ala Gly 420 425 430 Lys Ser Thr Leu Leu Asn Ile Leu Ser Gly Leu Ser Val Ser Thr Glu 435 440 445 Gly Ser Ala Thr Ile Tyr Asn Thr Gln Leu Ser Glu Ile Thr Asp Met 450 455 460 Glu Glu Ile Arg Lys Asn Ile Gly Phe Cys Pro Gln Phe Asn Phe Gln 465 470 475 480 Phe Asp Phe Leu Thr Val Arg Glu Asn Leu Arg Val Phe Ala Lys Ile 485 490 495 Lys Gly Ile Gln Pro Lys Glu Val Glu Gln Glu Val Lys Arg Ile Ile 500 505 510 Met Glu Leu Asp Met Gln Ser Ile Gln Asp Ile Ile Ala Lys Lys Leu 515 520 525 Ser Gly Gly Gln Lys Arg Lys Leu Thr Leu Gly Ile Ala Ile Leu Gly 530 535 540 Asp Pro Gln Val Leu Leu Leu Asp Glu Pro Thr Ala Gly Leu Asp Pro 545 550 555 560 Phe Ser Arg His Arg Val Trp Ser Leu Leu Lys Glu His Lys Val Asp 565 570 575 Arg Leu Ile Leu Phe Ser Thr Gln Phe Met Asp Glu Ala Asp Ile Leu 580 585 590 Ala Asp Arg Lys Val Phe Leu Ser Asn Gly Lys Leu Lys Cys Ala Gly 595 600 605 Ser Ser Leu Phe Leu Lys Arg Lys Trp Gly Ile Gly Tyr His Leu Ser 610 615 620 Leu His Arg Asn Glu Met Cys Asp Thr Glu Lys Ile Thr Ser Leu Ile 625 630 635 640 Lys Gln His Ile Pro Asp Ala Lys Leu Thr Thr Glu Ser Glu Glu Lys 645 650 655 Leu Val Tyr Ser Leu Pro Leu Glu Lys Thr Asn Lys Phe Pro Asp Leu 660 665 670 Tyr Ser Asp Leu Asp Lys Cys Ser Asp Gln Gly Ile Arg Asn Tyr Ala 675 680 685 Val Ser Val Thr Ser Leu Asn Glu Val Phe Leu Asn Leu Glu Gly Lys 690 695 700 Ser Ala Ile Asp Glu Pro Asp Phe Asp Ile Gly Lys Gln Glu Lys Ile 705 710 715 720 His Val Thr Arg Asn Thr Gly Asp Glu Ser Glu Met Glu Gln Val Leu 725 730 735 Cys Ser Leu Pro Glu Thr Arg Lys Ala Val Ser Ser Ala Ala Leu Trp 740 745 750 Arg Arg Gln Ile Tyr Ala Val Ala Thr Leu Arg Phe Leu Lys Leu Arg 755 760 765 Arg Glu Arg Arg Ala Leu Leu Cys Leu Leu Leu Val Leu Gly Ile Ala 770 775 780 Phe Ile Pro Ile Ile Leu Glu Lys Ile Met Tyr Lys Val Thr Arg Glu 785 790 795 800 Thr His Cys Trp Glu Phe Ser Pro Ser Met Tyr Phe Leu Ser Leu Glu 805 810 815 Gln Ile Pro Lys Thr Pro Leu Thr Ser Leu Leu Ile Val Asn Asn Thr 820 825 830 Gly Ser Asn Ile Glu Asp Leu Val His Ser Leu Lys Cys Gln Asp Ile 835 840 845 Val Leu Glu Ile Asp Asp Phe Arg Asn Arg Asn Gly Ser Asp Asp Pro 850 855 860 Ser Tyr Asn Gly Ala Ile Ile Val Ser Gly Asp Gln Lys Asp Tyr Arg 865 870 875 880 Phe Ser Val Ala Cys Asn Thr Lys Lys Leu Asn Cys Phe Pro Val Leu 885 890 895 Met Gly Ile Val Ser Asn Ala Leu Met Gly Ile Phe Asn Phe Thr Glu 900 905 910 Leu Ile Gln Thr Glu Ser Thr Ser Phe Ser Arg Asp Asp Ile Val Leu 915 920 925 Asp Leu Gly Phe Ile Asp Gly Ser Ile Phe Leu Leu Leu Ile Thr Asn 930 935 940 Cys Val Ser Pro Phe Ile Gly Met Ser Ser Ile Ser Asp Tyr Lys Lys 945 950 955 960 Asn Val Gln Ser Gln Leu Trp Ile Ser Gly Leu Trp Pro Ser Ala Tyr 965 970 975 Trp Cys Gly Gln Ala Leu Val Asp Ile Pro Leu Tyr Phe Leu Ile Leu 980 985 990 Phe Ser Ile His Leu Ile Tyr Tyr Phe Ile Phe Leu Gly Phe Gln Leu 995 1000 1005 Ser Trp Glu Leu Met Phe Val Leu Val Val Cys Ile Ile Gly Cys Ala 1010 1015 1020 Val Ser Leu Ile Phe Leu Thr Tyr Val Leu Ser Phe Ile Phe Arg Lys 1025 1030 1035 1040 Trp Arg Lys Asn Asn Gly Phe Trp Ser Phe Gly Phe Phe Ile Ile Leu 1045 1050 1055 Ile Cys Val Ser Thr Ile Met Val Ser Thr Gln Tyr Glu Lys Leu Asn 1060 1065 1070 Leu Ile Leu Cys Met Ile Phe Ile Pro Ser Phe Thr Leu Leu Gly Tyr 1075 1080 1085 Val Met Leu Leu Ile Gln Leu Asp Phe Met Arg Asn Leu Asp Ser Leu 1090 1095 1100 Asp Asn Arg Ile Asn Glu Val Asn Lys Thr Ile Leu Leu Thr Thr Leu 1105 1110 1115 1120 Ile Pro Tyr Leu Gln Ser Val Ile Phe Leu Phe Val Ile Arg Cys Leu 1125 1130 1135 Glu Met Lys Tyr Gly Asn Glu Ile Met Asn Lys Asp Pro Val Phe Arg 1140 1145 1150 Ile Ser Pro Arg Ser Arg Glu Thr His Pro Asn Pro Glu Glu Pro Glu 1155 1160 1165 Glu Glu Asp Glu Asp Val Gln Ala Glu Arg Val Gln Ala Ala Asn Ala 1170 1175 1180 Leu Thr Ala Pro Asn Leu Glu Glu Glu Pro Val Ile Thr Ala Ser Cys 1185 1190 1195 1200 Leu His Lys Glu Tyr Tyr Glu Thr Lys Lys Ser Cys Phe Ser Thr Arg 1205 1210 1215 Lys Lys Lys Ile Ala Ile Arg Asn Val Ser Phe Cys Val Lys Lys Gly 1220 1225 1230 Glu Val Leu Gly Leu Leu Gly His Asn Gly Ala Gly Lys Ser Thr Ser 1235 1240 1245 Ile Lys Met Ile Thr Gly Cys Thr Lys Pro Thr Ala Gly Val Val Val 1250 1255 1260 Leu Gln Gly Ser Arg Ala Ser Val Arg Gln Gln His Asp Asn Ser Leu 1265 1270 1275 1280 Lys Phe Leu Gly Tyr Cys Pro Gln Glu Asn Ser Leu Trp Pro Lys Leu 1285 1290 1295 Thr Met Lys Glu His Leu Glu Leu Tyr Ala Ala Val Lys Gly Leu Gly 1300 1305 1310 Lys Glu Asp Ala Ala Leu Ser Ile Ser Arg Leu Val Glu Ala Leu Lys 1315 1320 1325 Leu Gln Glu Gln Leu Lys Ala Pro Val Lys Thr Leu Ser Glu Gly Ile 1330 1335 1340 Lys Arg Lys Leu Cys Phe Val Leu Ser Ile Leu Gly Asn Pro Ser Val 1345 1350 1355 1360 Val Leu Leu Asp Glu Pro Phe Thr Gly Met Asp Pro Glu Gly Gln Gln 1365 1370 1375 Gln Met Trp Gln Ile Leu Gln Ala Thr Val Lys Asn Lys Glu Arg Gly 1380 1385 1390 Thr Leu Leu Thr Thr His Tyr Met Ser Glu Ala Glu Ala Val Cys Asp 1395 1400 1405 Arg Met Ala Met Met Val Ser Gly Thr Leu Arg Cys Ile Gly Ser Ile 1410 1415 1420 Gln His Leu Lys Asn Lys Phe Gly Arg Asp Tyr Leu Leu Glu Ile Lys 1425 1430 1435 1440 Met Lys Glu Pro Thr Gln Val Glu Ala Leu His Thr Glu Ile Leu Lys 1445 1450 1455 Leu Phe Pro Gln Ala Ala Trp Gln Glu Arg Tyr Ser Ser Leu Met Ala 1460 1465 1470 Tyr Lys Leu Pro Val Glu Asp Val His Pro Leu Ser Arg Ala Phe Phe 1475 1480 1485 Lys Leu Glu Ala Met Lys Gln Thr Phe Asn Leu Glu Glu Tyr Ser Leu 1490 1495 1500 Ser Gln Ala Thr Leu Glu Gln Val Phe Leu Glu Leu Cys Lys Glu Gln 1505 1510 1515 1520 Glu Leu Gly Asn Val Asp Asp Lys Ile Asp Thr Thr Val Glu Trp Lys 1525 1530 1535 Leu Leu Pro Gln Glu Asp Pro 1540 9 130 DNA Homo sapiens 9 ctgctggagt aggcacccat ttaaagaaaa aatgaagaag cagcaataaa gaagttgtaa 60 tcgttaccta gacaaacaga gaactggttt tgacagtgtt tctagagtgc tttttattat 120 tttcctgaca 130 10 141 DNA Homo sapiens 10 gttgtgttcc accatgatta ctttctcctt cagcgaatag gctaaatgaa tatgaaacag 60 aaaagcgtgt atcagcaaac caaagcactt ctgtgcaaga attttcttaa gaaatggagg 120 atgaaaagag agagcttatt g 141 11 205 DNA Homo sapiens 11 gaatggggcc tctcaatact tctaggactg tgtattgctc tgttttccag ttccatgaga 60 aatgtccagt ttcctggaat ggctcctcag aatctgggaa gggtagataa atttaatagc 120 tcttctttaa tggttgtgta tacaccaata tctaatttaa cccagcagat aatgaataaa 180 acagcacttg ctcctctttt gaaag 205 12 159 DNA Homo sapiens 12 gaacaagtgt cattggggca ccaaataaaa cacacatgga cgaaatactt ctggaaaatt 60 taccatatgc tatgggaatc atctttaatg aaactttctc ttataagtta atatttttcc 120 agggatataa cagtccactt tggaaagaag atttctcag 159 13 104 DNA Homo sapiens 13 ctcattgctg ggatggatat ggtgagtttt catgtacatt gaccaaatac tggaatagag 60 gatttgtggc tttacaaaca gctattaata ctgccattat agaa 104 14 227 DNA Homo sapiens 14 atcacaacca atcaccctgt gatggaggag ttgatgtcag ttactgctat aactatgaag 60 acattacctt tcataactaa aaatcttctt cacaatgaga tgtttatttt attcttcttg 120 cttcatttct ccccacttgt atattttata tcactcaatg taacaaaaga gagaaaaaag 180 tctaagaatt tgatgaaaat gatgggtctc caagattcag cattctg 227 15 142 DNA Homo sapiens 15 gctctcctgg ggtctaatct atgctggctt catctttatt atttccatat tcattacaat 60 tatcataaca ttcacccaaa ttatagtcat gactggcttc atggtcatat ttatactctt 120 ttttttatat ggcttatctt tg 142 16 186 DNA Homo sapiens 16 gtagctttgg tgttcctgat gagtgtgctg ttaaagaaag ctgtcctcac caatttggtt 60 gtgtttctcc ttaccctctt ttggggatgt ctgggattca ctgtatttta tgaacaactt 120 ccttcatctc tggagtggat tttgaatatt tgtagccctt ttgcctttac tactggaatg 180 attcag 186 17 148 DNA Homo sapiens 17 attatcaaac tggattataa cttgaatggt gtaatttttc ctgacccttc aggagactca 60 tatacaatga tagcaacttt ttctatgttg cttttggatg gtctcatcta cttgctattg 120 gcattatact ttgacaaaat tttaccct 148 18 169 DNA Homo sapiens 18 atggagatga gcgccattat tctcctttat ttttcttgaa ttcatcatct tgtttccaac 60 accaaaggac taatgctaag gttattgaga aagaaatcga tgctgagcat ccctctgatg 120 attattttga accagtagct cctgaattcc aaggaaaaga agccatcag 169 19 59 DNA Homo sapiens 19 aatcagaaat gttaagaagg aatataaagg aaaatctgga aaagtggaag cattgaaag 59 20 111 DNA Homo sapiens 20 gcttgctctt tgacatatat gaaggtcaaa tcacggcaat cctgggtcac agtggagctg 60 gcaaatcttc actgctaaat attcttaatg gattgtctgt tccaacagaa g 111 21 176 DNA Homo sapiens 21 gatcagttac catctataat aaaaatctct ctgaaatgca agacttggag gaaatcagaa 60 agataactgg cgtctgtcct caattcaatg ttcaatttga catactcacc gtgaaggaaa 120 acctcagcct gtttgctaaa ataaaaggga ttcatctaaa ggaagtggaa caagag 176 22 120 DNA Homo sapiens 22 gtacaacgaa tattattgga attggacatg caaaacattc aagataacct tgctaaacat 60 ttaagtgaag gacagaaaag aaagctgact tttgggatta ccattttagg agatcctcaa 120 23 139 DNA Homo sapiens 23 attttgcttt tagatgaacc aactactgga ttggatccct tttccagaga tcaagtgtgg 60 agcctcctga gagagcgtag agcagatcat gtgatccttt tcagtaccca gtccatggat 120 gaggctgaca tcctggctg 139 24 91 DNA Homo sapiens 24 atagaaaagt gatcatgtcc aatgggagac tgaagtgtgc aggttcttct atgtttttga 60 aaagaaggtg gggtcttgga tatcacctaa g 91 25 140 DNA Homo sapiens 25 tttacatagg aatgaaatat gtaacccaga acaaataaca tccttcatta ctcatcacat 60 ccccgatgct aaattaaaaa cagaaaacaa agaaaagctt gtatatactt tgccactgga 120 aaggacaaat acatttccag 140 26 117 DNA Homo sapiens 26 atcttttcag tgatctggat aagtgttctg accagggagt gacaggttat gacatttcca 60 tgtcaactct aaatgaagtc tttatgaaac tggaaggaca gtcaactatc gaacaag 117 27 184 DNA Homo sapiens 27 atttcgaaca agtggagatg ataagagact cagaaagcct caatgaaatg gagctggctc 60 actcttcctt ctctgaaatg cagacagctg tgagtgacat gggcctctgg agaatgcaag 120 tctttgccat ggcacggctc cgtttcttaa agttaaaacg tcaaactaaa gtgttattga 180 ccct 184 28 167 DNA Homo sapiens 28 attattggta tttggaatcg caatattccc tttgattgtt gaaaatataa tatatgctat 60 gttaaatgaa aagatcgatt gggaatttaa aaacgaattg tattttctct ctcctggaca 120 acttccccag gaaccccgta ccagcctgtt gatcatcaat aacacag 167 29 134 DNA Homo sapiens 29 aatcaaatat tgaagatttt ataaaatcac tgaagcatca aaatatactt ttggaagtag 60 atgactttga aaacagaaat ggtactgatg gcctctcata caatggagct atcatagttt 120 ctggtaaaca aaag 134 30 138 DNA Homo sapiens 30 gattatagat tttcagttgt gtgtaatacc aagagattgc actgttttcc aattcttatg 60 aatattatca gcaatgggct acttcaaatg tttaatcaca cacaacatat tcgaattgag 120 tcaagcccat ttcctctt 138 31 108 DNA Homo sapiens 31 agccacatag gactctggac tgggttgccg gatggttcct ttttcttatt tttggttcta 60 tgtagcattt ctccttatat caccatgggc agcatcagtg attacaag 108 32 174 DNA Homo sapiens 32 aaaaatgcta agtcccagct atggatttca ggcctctaca cttctgctta ctggtgtggg 60 caggcactag tggacgtcag cttcttcatt ttaattctcc ttttaatgta tttaattttc 120 tacatagaaa acatgcagta ccttcttatt acaagccaaa ttgtgtttgc tttg 174 33 114 DNA Homo sapiens 33 gttatagtta ctcctggtta tgcagcttct cttgtcttct tcatatatat gatatcattt 60 atttttcgca aaaggagaaa aaacagtggc ctttggtcat tttacttctt tttt 114 34 120 DNA Homo sapiens 34 gcctccacca tcatgttttc catcacttta atcaatcatt ttgacctaag tatattgatt 60 accaccatgg tattggttcc ttcatatacc ttgcttggat ttaaaacttt tttggaagtg 120 35 78 DNA Homo sapiens 35 agagaccagg agcactacag agaatttcca gaggcaaatt ttgaattgag tgccactgat 60 tttctagtct gcttcata 78 36 92 DNA Homo sapiens 36 ccctactttc agactttgct attcgttttt gttctaagat gcatggaact aaaatgtgga 60 aagaaaagaa tgcgaaaaga tcctgttttc ag 92 37 121 DNA Homo sapiens 37 aatttccccc caaagtagag atgctaagcc aaatccagaa gaacccatag atgaagatga 60 agatattcaa acagaaagaa taagaacagc cactgctctg accacttcaa tcttagatga 120 g 121 38 118 DNA Homo sapiens 38 aaacctgtta taattgccag ctgtctacac aaagaatatg caggccagaa gaaaagttgc 60 ttttcaaaga ggaagaagaa aatagcagca agaaatatct ctttctgtgt tcaagaag 118 39 92 DNA Homo sapiens 39 gtgaaatttt gggattgcta ggacccagtg gtgctggaaa aagttcatct attagaatga 60 tatctgggat cacaaagcca actgctggag ag 92 40 155 DNA Homo sapiens 40 gtggaactga aaggctgcag ttcagttttg ggccacctgg ggtactgccc tcaagagaac 60 gtgctgtggc ccatgctgac gttgagggaa cacctggagg tgtatgctgc cgtcaagggg 120 ctcaggaaag cggacgcgag gctcgccatc gcaag 155 41 76 DNA Homo sapiens 41 attagtgagt gctttcaaac tgcatgagca gctgaatgtt cctgtgcaga aattaacagc 60 aggaatcacg agaaag 76 42 95 DNA Homo sapiens 42 ttgtgttttg tgctgagcct cctgggaaac tcacctgtct tgctcctgga tgaaccatct 60 acgggcatag accccacagg gcagcagcaa atgtg 95 43 120 DNA Homo sapiens 43 gcaggcaatc caggcagtcg ttaaaaacac agagagaggt gtcctcctga ccacccataa 60 cctggctgag gcggaagcct tgtgtgaccg tgtggccatc atggtgtctg gaaggcttag 120 44 141 DNA Homo sapiens 44 atgcattggc tccatccaac acctgaaaaa caaacttggc aaggattaca ttctagagct 60 aaaagtgaag gaaacgtctc aagtgacttt ggtccacact gagattctga agcttttccc 120 acaggctgca gggcaggaaa g 141 45 80 DNA Homo sapiens 45 gtattcctct ttgttaacct ataagctgcc cgtggcagac gtttaccctc tatcacagac 60 ctttcacaaa ttagaagcag 80 46 56 DNA Homo sapiens 46 tgaagcataa ctttaacctg gaagaataca gcctttctca gtgcacactg gagaag 56 47 369 DNA Homo sapiens 47 gtattcttag agctttctaa agaacaggaa gtaggaaatt ttgatgaaga aattgataca 60 acaatgagat ggaaactcct ccctcattca gatgaacctt aaaacctcaa acctagtaat 120 tttttgttga tctcctataa acttatgttt tatgtaataa ttaatagtat gtttaatttt 180 aaagatcatt taaaattaac atcaggtata ttttgtaaat ttagttaaca aatacataaa 240 ttttaaaatt attcttcctc tcaaacatag gggtgatagc aaacctgtga taaaggcaat 300 acaaaatatt agtaaagtca cccaaagagt caggcactgg gtattgtgga aataaaacta 360 tataaactt 369 48 130 DNA Homo sapiens 48 attcacaatg aatgtgaaat taaaagcatg atgtagtagt gacccaaaag gaatgtgaat 60 tctcctccag aacatgcaga gacccatgga tgaactgtgt ttctagattt ttcctccagc 120 tttcctgaga 130 49 109 DNA Homo sapiens 49 gaaacaggtc aaaatgagca agagacgcat gagcgtgggt cagcaaacat gggctcttct 60 ctgcaagaac tgtctcaaaa aatggagaat gaaaagacag accttgttg 109 50 208 DNA Homo sapiens 50 gaatggctct tttcatttct tctggtactg tttctgtacc tatttttctc caatttacat 60 caagttcatg acactcctca aatgtcttca atggatctgg gacgtgtaga tagttttaat 120 gatactaatt atgttattgc atttgcacct gaatccaaaa ctacccaaga gataatgaac 180 aaagtggctt cagccccatt cctaaaag 208 51 165 DNA Homo sapiens 51 gaagaacaat catggggtgg cctgatgaaa aaagcatgga tgaattggat ttgaactatt 60 caatagacgc agtgagagtc atctttactg ataccttctc ctaccatttg aagttttctt 120 ggggacatag aatccccatg atgaaagagc acagagacca ttcag 165 52 104 DNA Homo sapiens 52 ctcactgtca agcagtgaat gaaaaaatga agtgtgaagg ttcagagttc tgggagaaag 60 gctttgtagc ttttcaagct gccattaatg ctgctatcat agaa 104 53 227 DNA Homo sapiens 53 atcgcaacaa atcattcagt gatggaacag ctgatgtcag ttactggtgt acatatgaag 60 atattacctt ttgttgccca aggaggagtt gcaactgatt ttttcatttt cttttgcatt 120 atttcttttt ctacatttat atactatgta tcagtcaatg ttacacaaga aagacaatac 180 attacgtcat tgatgacaat gatgggactc cgagagtcag cattctg 227 54 142 DNA Homo sapiens 54 gctttcctgg ggtttgatgt atgctggctt catccttatc atggccactt taatggctct 60 tattgtaaaa tctgcacaaa ttgtcgtcct gactggtttt gtgatggtct tcaccctctt 120 tctcctctat ggcctgtctt tg 142 55 186 DNA Homo sapiens 55 ataactttag ctttcctgat gagtgtgttg ataaagaaac ctttccttac gggcttggtt 60 gtgtttctcc ttattgtctt ttgggggatc ctgggattcc cagcattgta tacacatctt 120 cctgcatttt tggaatggac tttgtgtctt cttagcccct ttgccttcac tgttgggatg 180 gcccag 186 56 148 DNA Homo sapiens 56 cttatacatt tggactatga tgtgaattct aatgcccact tggattcttc acaaaatcca 60 tacctcataa tagctactct tttcatgttg gtttttgaca cccttctgta tttggtattg 120 acattatatt ttgacaaaat tttgcccg 148 57 169 DNA Homo sapiens 57 ctgaatatgg acatcgatgt tctcccttgt ttttcctgaa atcctgtttt tggtttcaac 60 acggaagggc taatcatgtg gtccttgaga atgaaacaga ttctgatcct acacctaatg 120 actgttttga accagtgtct ccagaattct gtgggaagga agccatcag 169 58 59 DNA Homo sapiens 58 aatcaaaaat cttaaaaaag aatatgcagg gaagtgtgag agagtagaag ctttgaaag 59 59 111 DNA Homo sapiens 59 gtgtggtgtt tgacatatat gaaggccaga tcactgccct ccttggtcac agtggagctg 60 gaaaaactac cctgttaaac atacttagtg ggttgtcagt tccaacatca g 111 60 176 DNA Homo sapiens 60 gttcagtcac tgtctataat cacacacttt caagaatggc tgatatagaa aatatcagca 60 agttcactgg attttgtcca caatccaatg tgcaatttgg atttctcact gtgaaagaaa 120 acctcaggct gtttgctaaa ataaaaggga ttttgccaca tgaagtggag aaagag 176 61 120 DNA Homo sapiens 61 gtacaacgag ttgtacagga attagaaatg gaaaatattc aagacatcct tgctcaaaac 60 ttaagtggtg gacaaaatag gaaactaact tttgggattg ccattttagg agatcctcaa 120 62 139 DNA Homo sapiens 62 gttttgctat tggatgaacc gactgctgga ttggatcctc tttcaaggca ccgaatatgg 60 aatctcctga aagaggggaa atcagacaga gtaattctct tcagcaccca gtttatagat 120 gaggctgaca ttctggcgg 139 63 91 DNA Homo sapiens 63 acaggaaggt gttcatatcc aatgggaagc tgaagtgtgc aggctcttct ctgttcctta 60 agaagaaatg gggcataggc taccatttaa g 91 64 140 DNA Homo sapiens 64 tttgcatctg aatgaaaggt gtgatccaga gagtataaca tcactggtta agcagcacat 60 ctctgatgcc aaattgacag cacaaagtga agaaaaactt gtatatattt tgcctttgga 120 aaggacaaac aaatttccag 140 65 120 DNA Homo sapiens 65 aactttacag ggatcttgat agatgttcta accaaggcat tgaggattat ggtgtttcca 60 taacaacttt gaatgaggtg tttctgaaat tagaaggaaa atcaactatt gatgaatcag 120 66 199 DNA Homo sapiens 66 atattggaat ttggggacaa ttacaaactg atggggcaaa agatatagga agccttgttg 60 agctggaaca agttttgtct tccttccacg aaacaaggaa aacaatcagt ggcgtggcgc 120 tctggaggca gcaggtctgt gcaatagcaa aagttcgctt cctaaagtta aagaaagaaa 180 gaaaaagcct gtggactat 199 67 167 DNA Homo sapiens 67 attattgctt tttggtatta gctttatccc tcaacttttg gaacatctat tctacgagtc 60 atatcagaaa agttacccgt gggaactgtc tccaaataca tacttcctct caccaggaca 120 acaaccacag gatcctctga cccatttact ggtcatcaat aagacag 167 68 134 DNA Homo sapiens 68 ggtcaaccat tgataacttt ttacattcac tgaggcgaca gaacatagct atagaagtgg 60 atgcctttgg aactagaaat ggcacagatg acccatctta caatggtgct atcattgtgt 120 caggtgatga aaag 134 69 138 DNA Homo sapiens 69 gatcacagat tttcaatagc atgtaataca aaacggctga attgctttcc tgtcctcctg 60 gatgtcatta gcaatggact acttggaatt tttaattcgt cagaacacat tcagactgac 120 agaagcacat tttttgaa 138 70 108 DNA Homo sapiens 70 gagcatatgg attatgagta tgggtaccga agtaacacct tcttctggat accgatggca 60 gcctctttca ctccatacat tgcaatgagc agcattggtg actacaaa 108 71 174 DNA Homo sapiens 71 aaaaaagctc attcccagct acggatttca ggcctctacc cttctgcata ctggtttggc 60 caagcactgg tggatgtttc cctgtacttt ttgatcctcc tgctaatgca aataatggat 120 tatattttta gcccagagga gattatattt ataattcaaa acctgttaat tcaa 174 72 114 DNA Homo sapiens 72 atcctgtgta gtattggcta tgtctcatct cttgttttct tgacatatgt gatttcattc 60 atttttcgca atgggagaaa aaatagtggc atttggtcat ttttcttctt aatt 114 73 120 DNA Homo sapiens 73 gtggtcatct tctcgatagt tgctactgat ctaaatgaat atggatttct agggctattt 60 tttggcacca tgttaatacc tcccttcaca ttgattggct ctctattcat tttttctgag 120 74 69 DNA Homo sapiens 74 atttctcctg attccatgga ttacttagga gcttcagaat ctgaaattgt atacctggca 60 ctgctaata 69 75 92 DNA Homo sapiens 75 ccttaccttc attttctcat ttttcttttc attctgcgat gcctagaaat gaactgcagg 60 aagaaactaa tgagaaagga tcctgtgttc ag 92 76 121 DNA Homo sapiens 76 aatttctcca agaagcaacg ctatttttcc aaacccagaa gagcctgaag gagaggagga 60 agatatccag atggaaagaa tgagaacagt gaatgctatg gctgtgcgag actttgatga 120 g 121 77 118 DNA Homo sapiens 77 acacccgtca tcattgccag ctgtctacgg aaggaatatg caggcaaaaa gaaaaattgc 60 ttttctaaaa ggaagaaaac aattgccaca agaaatgtct ctttttgtgt taaaaaag 118 78 92 DNA Homo sapiens 78 gtgaagttat aggactgtta ggacacaatg gagctggtaa aagtacaact attaagatga 60 taactggaga cacaaaacca actgcaggac ag 92 79 161 DNA Homo sapiens 79 gtgattttga aagggagcgg tggaggggaa cccctgggct tcctggggta ctgccctcag 60 gagaatgcgc tgtggcccaa cctgacagtg aggcagcacc tggaggtgta cgctgccgtg 120 aaaggtctca ggaaagggga cgcaatgatc gccatcacac g 161 80 76 DNA Homo sapiens 80 gttagtggat gcgctcaagc tgcaggacca gctgaaggct cccgtgaaga ccttgtcaga 60 gggaataaag cgaaag 76 81 95 DNA Homo sapiens 81 ctgcgctttg tgctgagcat cctggggaac ccgtcagtgg tgcttctgga tgagccgtcg 60 accgggatgg accccgaggg gcagcagcaa atgtg 95 82 120 DNA Homo sapiens 82 gcaggtgatt cgggccacct ttagaaacac ggagaggggc gccctcctga ccacccacta 60 catggcagag gctgaggcgg tgtgtgaccg agtggccatc atggtgtcag gaaggctgag 120 83 141 DNA Homo sapiens 83 atgtattggt tccatccaac acctgaaaag caaatttggc aaagactacc tgctggagat 60 gaagctgaag aacctggcac aaatggagcc cctccatgca gagatcctga ggcttttccc 120 ccaggctgct cagcaggaaa g 141 84 80 DNA Homo sapiens 84 gttctcctcc ctgatggtct ataagttgcc tgttgaggat gtgcgacctt tatcacaggc 60 tttcttcaaa ttagagatag 80 85 56 DNA Homo sapiens 85 ttaaacagag tttcgacctg gaggagtaca gcctctcaca gtctaccctg gagcag 56 86 1062 DNA Homo sapiens 86 gttttcctgg agctctccaa ggagcaggag ctgggtgatc ttgaagagga ctttgatccc 60 tcggtgaagt ggaaactcct cctgcaggaa gagccttaaa gctccaaata ccctatatct 120 ttctttaatc ctgtgactct tttaaagata atattttata gccttaatat gccttatatc 180 agaggtggta caaaatgcat ttgaaactca tgcaataatt atcctcagta gtatttctta 240 cagtgagaca acaggcaatg tcagtgaggg cgatcgtagg gcataagcct aagccatacc 300 atgcagcctt tgtgccagca accaaatccc atgtttccta ctgtgttaag tttaaaaatg 360 catttattat agaattgtct acatttctga ggatgtcatg gagaatgctt aattttcttt 420 ctctgaactt caaaatatta aatattttct tatttttttg attaaagtat aaattaagac 480 accctattga cttccgggta aggggagtca attgattacc cagcagcaca gtatttgctt 540 tttataattc cctttttaaa tacttgttct taattgactg gttttccttt tctgtcattt 600 ttcagagttt agattgtgag tccatgtttt gtctgttgtg cctataaagg aaatttgaaa 660 tctgtatcat tctactataa agacacatgc acacgtatgt ttattgcagc actgtttaca 720 atagcaaaga cttggaacca accaaaatac ccacaaatga tagaccggat aaagaaaacg 780 tgacacatat acaccatgga atactatgca gccatagaaa aggatgagtt catattcttc 840 acagggacat ggatgaagct ggaaaccatc atcctcagca aactaacaca ggaacagaaa 900 accaaacacc gcatgttctc actcataagt gggaattgaa caatgagaat acatggacac 960 agggagggga acaccacacc ctggggcctg ttggggggat gggggctagg ggagggatag 1020 cattaggaga aatacctgat gtagatgatg ggttgatggg tg 1062 87 287 DNA Homo sapiens 87 aattaatttt acttaggata agtgttgtta ttattgtttt tattgttgtt ctgttagtta 60 ctcaaaactt cattctaatt gtgccctgag tttgttaaaa taccatactg tatttttgtg 120 taacatgtaa ataggcatta atttttgaga aatagaaatg tttatcctta atgtattttt 180 aatttgctaa cattgatttt ttattttctt tcctgaaata gcttatttcc taaaatgaaa 240 gaatttattc tcagatgaat aatttttata tcagctattc ttatcag 287 88 280 DNA Homo sapiens 88 agcaataaac aaataccaat gatgcgctca gccaacaatt cattacactc tctgaagagt 60 aactggacaa ggagaaaaac atagggaaaa aaccaacaga atttgttggc atgttctaca 120 cacagaccat ggcttttcag aagccaagct gaataaaaac agttttaaaa gaggcaacca 180 tttgtagagg agtccttgaa ggattcttca ttgttttctt ggacaaaaag agaccagtgg 240 atccaagtgc ttcaaatact tctctcttat tttcttaact 280 89 141 DNA Homo sapiens 89 ctattgctct gcaatattta ctttaccctg ttaatgaaca ggacaaaatg gttaaaaaag 60 agataagcgt gcgtcaacaa attcaggctc ttctgtacaa gaattttctt aaaaaatgga 120 gaataaaaag agagtttatt g 141 90 205 DNA Homo sapiens 90 gaatggacaa taacattgtt tctagggcta tatttgtgca tcttttcgga acacttcaga 60 gctacccgtt ttcctgaaca acctcctaaa gtcctgggaa gcgtggatca gtttaatgac 120 tctggcctgg tagtggcata tacaccagtc agtaacataa cacaaaggat aatgaataag 180 atggccttgg cttcctttat gaaag 205 91 165 DNA Homo sapiens 91 gaagaacagt cattgggaca ccagatgaag agaccatgga tatagaactt ccaaaaaaat 60 accatgaaat ggtgggagtt atatttagtg atactttctc atatcgcctg aagtttaatt 120 ggggatatag aatcccagtt ataaaggagc actctgaata cacag 165 92 104 DNA Homo sapiens 92 aacactgttg ggccatgcat ggtgaaattt tttgttactt ggcaaagtac tggctaaaag 60 ggtttgtagc ttttcaagct gcaattaatg ctgcaattat agaa 104 93 227 DNA Homo sapiens 93 gtcacaacaa atcattctgt aatggaggag ttgacatcag ttattggaat aaatatgaag 60 ataccacctt tcatttctaa gggagaaatt atgaatgaat ggtttcattt tacttgctta 120 gtttctttct cttcttttat atactttgca tcattaaatg ttgcaaggga aagaggaaaa 180 tttaagaaac tgatgacagt aatgggtctc cgagagtcag cattctg 227 94 142 DNA Homo sapiens unsure 11 n=unknown, may be a or g or c or t 94 gctctcctgg ngattgacat acatttgctt catcttcatt atgtccattt ttatggctct 60 ggtcataaca tcaatctcaa ttgtatttca tactggcttc atggtgatat tcacactcta 120 tagcttatat ggcctttctt tg 142 95 186 DNA Homo sapiens 95 atagcattgg ctttcctcat gagtgtttta ataaggaaac ctatgctcgc tggtttggct 60 ggatttctct tcactgtatt ttggggatgt ctgggattca ctgtgttata cagacaactt 120 cctttatctt tgggatgggt attaagtctt cttagccctt ttgccttcac tgctggaatg 180 gcccag 186 96 148 DNA Homo sapiens 96 gttacacacc tggataatta cttaagtggt gttatttttc ctgatccctc tggggattca 60 tacaaaatga tagccacttt tttcattttg gcatttgata ctcttttcta tttgatattc 120 acattatatt ttgagcgagt tttacctg 148 97 169 DNA Homo sapiens 97 ataaagatgg ccatggggat tctccattat ttttccttaa gtcctcattt tggtccaaac 60 atcaaaatac tcatcatgaa atctttgaga atgaaataaa tcctgagcat tcctctgatg 120 attcttttga accggtgtct ccagaattcc atggaaaaga agccataag 169 98 59 DNA Homo sapiens 98 aatcagaaat gttataaaag aatataatgg aaagactgga aaagtagaag cattgcaag 59 99 111 DNA Homo sapiens 99 gcatattttt tgacatatat gaaggacaga tcactgcaat acttgggcat aatggagctg 60 gtaaatcaac actgctaaac attcttagtg gattgtctgt ttctacagaa g 111 100 176 DNA Homo sapiens 100 gatcagccac tatttataat actcaactct ctgaaataac tgacatggaa gaaattagaa 60 agaatattgg attttgtcca cagttcaatt ttcaatttga cttcctcact gtgagagaaa 120 acctcagggt atttgctaaa ataaaaggga ttcagccaaa ggaagtggaa caagag 176 101 120 DNA Homo sapiens 101 gtaaaaagaa ttataatgga attagacatg caaagcattc aagacattat tgctaaaaaa 60 ttaagtggtg ggcagaagag aaaactaaca ctagggattg ccatcttagg agatcctcag 120 102 139 DNA Homo sapiens 102 gttttgctgc tagatgaacc aactgctgga ttggatccct tttcaagaca ccgagtgtgg 60 agcctcctga aggagcataa agtagaccga cttatcctct tcagtaccca attcatggat 120 gaggctgaca tcttggctg 139 103 91 DNA Homo sapiens 103 ataggaaagt atttctgtct aatgggaagt tgaaatgtgc aggatcatct ttgtttctga 60 agcgaaagtg gggtattgga tatcatttaa g 91 104 140 DNA Homo sapiens 104 tttacacagg aatgaaatgt gtgacacaga aaaaatcaca tcccttatta agcagcacat 60 tcctgatgcc aagttaacaa cagaaagtga agaaaaactt gtatatagtt tgcctttgga 120 aaaaacgaac aaatttccag 140 105 120 DNA Homo sapiens 105 atctttacag tgaccttgat aagtgttctg accagggcat aaggaattat gctgtttcag 60 tgacatctct gaatgaagta ttcttgaacc tagaaggaaa atcagcaatt gatgaaccag 120 106 199 DNA Homo sapiens 106 attttgacat tgggaaacaa gagaaaatac atgtgacaag aaatactgga gatgagtctg 60 aaatggaaca ggttctttgt tctcttcctg aaacaagaaa ggctgtcagt agtgcagctc 120 tctggagacg acaaatctat gcagtggcaa cacttcgctt cttaaagtta aggcgtgaaa 180 ggagagctct tttgtgttt 199 107 167 DNA Homo sapiens 107 gttactagta cttggaattg cttttatccc catcattcta gagaagataa tgtataaagt 60 aactcgtgaa actcattgtt gggagttttc acccagtatg tatttccttt ctctggaaca 120 aatcccgaag acgcctctta ccagcctgtt aatcgttaat aatacag 167 108 134 DNA Homo sapiens 108 gatcaaatat tgaagacctc gtgcattcac tgaagtgtca ggatatagtt ttggaaatag 60 atgactttag aaacagaaat ggctcagatg atccctccta caatggagcc atcatagtgt 120 ctggtgacca gaag 134 109 138 DNA Homo sapiens 109 gattacagat tttctgttgc gtgtaatacc aagaaattga attgttttcc tgttcttatg 60 ggaattgtta gcaatgccct tatgggaatt tttaacttca cggagcttat tcaaacggag 120 agcacttcat tttctcgt 138 110 108 DNA Homo sapiens 110 gatgacatag tgctggatct tggttttata gatgggtcca tatttttgtt gttgatcaca 60 aactgcgttt ctccttttat cggcatgagc agcatcagcg attataaa 108 111 171 DNA Homo sapiens 111 aaaaatgttc aatcccagtt atggatttca ggcctctggc cttcagcata ctggtgtgga 60 caggctctgg tggacattcc attatacttc ttgattctct tttcaataca tttaatttac 120 tacttcatat ttctgggatt ccagctttca tgggaactca tgtttgtttt g 171 112 114 DNA Homo sapiens 112 gtggtatgca taattggttg tgcagtttct cttatattcc tcacatatgt gctttcattc 60 atctttcgca agtggagaaa aaataatggc ttttggtctt ttggcttttt tatt 114 113 120 DNA Homo sapiens 113 atcttaatat gtgtatccac aattatggta tcaactcaat atgaaaaact caacttaatt 60 ttgtgcatga ttttcatacc ttccttcact ttgctggggt atgtcatgtt attgatccag 120 114 81 DNA Homo sapiens 114 ctcgacttta tgagaaactt ggacagtctg gacaatagaa taaatgaagt caataaaacc 60 attcttttaa caaccttaat a 81 115 92 DNA Homo sapiens 115 ccataccttc agagtgttat tttccttttt gtcataaggt gtctggaaat gaagtatgga 60 aatgaaataa tgaataaaga cccagttttc ag 92 116 121 DNA Homo sapiens 116 aatctctcca cggagtagag aaactcatcc caatccggaa gagcccgaag aagaagatga 60 agatgttcaa gctgaaagag tccaagcagc aaatgcactc actgctccaa acttggagga 120 g 121 117 118 DNA Homo sapiens 117 gaaccagtca taactgcaag ctgtttacac aaggaatatt atgagacaaa gaaaagttgc 60 ttttcaacaa gaaagaagaa aatagccatc agaaatgttt ccttttgtgt taaaaaag 118 118 92 DNA Homo sapiens 118 gtgaagtttt gggattacta ggacacaatg gagctggtaa aagtacttcc attaaaatga 60 taactgggtg cacaaagcca actgcaggag tg 92 119 179 DNA Homo sapiens 119 gtggtgttac aaggcagcag agcatcagta aggcaacagc atgacaacag cctcaagttc 60 ttggggtact gccctcagga gaactcactg tggcccaagc ttacaatgaa agagcacttg 120 gagttgtatg cagctgtgaa aggactgggc aaagaagatg ctgctctcag tatttcacg 179 120 76 DNA Homo sapiens 120 attggtggaa gctcttaagc tccaggaaca acttaaggct cctgtgaaaa ctctatcaga 60 gggaataaag agaaag 76 121 95 DNA Homo sapiens 121 ctgtgctttg tgctgagcat cctggggaac ccatcagtgg tgcttctaga tgagccgttc 60 accgggatgg accccgaggg gcagcagcaa atgtg 95 122 120 DNA Homo sapiens 122 gcagatactt caggctaccg ttaaaaacaa ggagaggggc accctcttga ccacccatta 60 catgtcagag gctgaggctg tgtgtgaccg tatggccatg atggtgtcag gaacgctaag 120 123 141 DNA Homo sapiens 123 gtgtattggt tccattcaac atctgaaaaa caagtttggt agagattatt tactagaaat 60 aaaaatgaaa gaacctaccc aggtggaagc tctccacaca gagattttga agcttttccc 120 acaggctgct tggcaggaaa g 141 124 80 DNA Homo sapiens 124 atattcctct ttaatggcgt ataagttacc tgtggaggat gtccaccctc tatctcgggc 60 ctttttcaag ttagaggcga 80 125 56 DNA Homo sapiens 125 tgaaacagac cttcaacctg gaggaataca gcctctctca ggctaccttg gagcag 56 126 769 DNA Homo sapiens 126 gtattcttag aactctgtaa agagcaggag ctgggaaatg ttgatgataa aattgataca 60 acagttgaat ggaaacttct cccacaggaa gacccttaaa atgaagaacc tcctaacatt 120 caattttagg tcctactaca ttgttagttt ccataattct acaagaatgt ttccttttac 180 ttcagttaac aaaagaaaac atttaataaa cattcaataa tgattacagt tttcattttt 240 aaaaatttag gatgaaggaa acaaggaaat atagggaaaa gtagtagaca aaattaacaa 300 aatcagacat gttattcatc cccaacatgg gtctattttg tgcttaaaaa taatttaaaa 360 atcatacaat attaggttgg ttttcggtta ttatcaataa agctaacact gagaacattt 420 tacaaataaa aatatgagtt ttttagcctg aacttcaaat gtatcagcta tttttaaaca 480 ttatttactc ggattctaat ttaatgtgac attgactata agaaggtctg ataaactgat 540 gaaatggcac agcataacat ttaattataa tgacattctg attataaaat aaatgcatgt 600 gaattttagt acatattgaa gttatatgga agaagatagc cataatctgt aagaaagtac 660 cgcagttaat attttcttta gccaacttat attcaatgta ttttttatgg atcctttttc 720 aaaggtagta tcagtaggca tagtcatttt ctgtatcttt tcacctcac 769 127 19 DNA Homo sapiens 127 cagtgactat gtatccgtg 19 128 19 DNA Homo sapiens 128 gatggtttct cctcacaac 19 129 19 DNA Homo sapiens 129 caccagacaa tgaggatga 19 130 19 DNA Homo sapiens 130 gctatattct tcaatggca 19 131 19 DNA Homo sapiens 131 cctagaagta gaccgcctt 19 132 19 DNA Homo sapiens 132 gttgtgagga gaaaccatc 19 133 19 DNA Homo sapiens 133 ctggatggtt tcagtcaca 19 134 19 DNA Homo sapiens 134 cagaaaagcc aatcgggtg 19 135 23 DNA Homo sapiens 135 ccaggtatat gttgtttaac cag 23 136 20 DNA Homo sapiens 136 gggtcagatt actgccttac 20 137 20 DNA Homo sapiens 137 gaacattgaa gaaccaacac 20 138 20 DNA Homo sapiens 138 gtaaggcagt aatctgaccc 20 139 18 DNA Homo sapiens 139 ggaaactgga cagaatgc 18 140 19 DNA Homo sapiens 140 ctaccctatt tcacatgcc 19 141 20 DNA Homo sapiens 141 gtttctccca taataacagc 20 142 20 DNA Homo sapiens 142 gctgttatta tgggagaaac 20 143 30 DNA Homo sapiens 143 agactacagt aacaaaagcc tagtgcagcc 30 144 30 DNA Homo sapiens 144 atccaatcct attagtgtga caaaggcttg 30 145 20 DNA Homo sapiens 145 tcagcaaacc aaagcacttc 20 146 27 DNA Homo sapiens 146 caagtgctgt tttattcatt atctgct 27 147 28 DNA Homo sapiens 147 gtacatgaaa actcaccata tccatccc 28 148 20 DNA Homo sapiens 148 tcattgctgg gatggatatg 20 149 19 DNA Homo sapiens 149 ccctgtgatg gaggagttg 19 150 21 DNA Homo sapiens 150 tgacatcaac tcctccatca c 21 151 20 DNA Homo sapiens 151 gaatgctgaa tcttggagac 20 152 20 DNA Homo sapiens 152 gattcagatt atcaaactgg 20 153 21 DNA Homo sapiens 153 tggtgtaatt tttcctgacc c 21 154 22 DNA Homo sapiens 154 aagggtcagg aaaaattaca cc 22 155 19 DNA Homo sapiens 155 ggaattcagg agctactgg 19 156 22 DNA Homo sapiens 156 gattgtctgt tccaacagaa gg 22 157 22 DNA Homo sapiens 157 ccacttcctt tagatgaatc cc 22 158 22 DNA Homo sapiens 158 aagtggaaca agaggtacaa cg 22 159 21 DNA Homo sapiens 159 atggtaatcc caaaagtcag c 21 160 22 DNA Homo sapiens 160 ggggatgtga tgagtaatga ag 22 161 22 DNA Homo sapiens 161 cttcattact catcacatcc cc 22 162 19 DNA Homo sapiens 162 acaacttccc caggaaccc 19 163 19 DNA Homo sapiens 163 gatcaacagg ctggtacgg 19 164 22 DNA Homo sapiens 164 caagaaaaat gctaagtccc ag 22 165 19 DNA Homo sapiens 165 tgcccacacc agtaagcag 19 166 22 DNA Homo sapiens 166 gaaaatcagt ggcactcaat tc 22 167 22 DNA Homo sapiens 167 tgccactgat tttctagtct gc 22 168 19 DNA Homo sapiens 168 ctgggatcac aaagccaac 19 169 20 DNA Homo sapiens 169 cctttcagtt ccacctctcc 20 170 21 DNA Homo sapiens 170 tccacactga gattctgaag c 21 171 19 DNA Homo sapiens 171 aatacctttc ctgccctgc 19 172 19 DNA Homo sapiens 172 gcctgactct ttgggtgac 19 173 19 DNA Homo sapiens 173 tgagcgtggg tcagcaaac 19 174 20 DNA Homo sapiens 174 gcaactcctc cttgggcaac 20 175 19 DNA Homo sapiens 175 tttgttgccc aaggaggag 19 176 22 DNA Homo sapiens 176 ggaaaaacaa gggagaacat cg 22 177 19 DNA Homo sapiens 177 gcccacttgg attcttcac 19 178 22 DNA Homo sapiens 178 ccacaccttt caaagcttct ac 22 179 22 DNA Homo sapiens 179 atgtggtcct tgagaatgaa ac 22 180 22 DNA Homo sapiens 180 actgtgaaag aaaacctcag gc 22 181 19 DNA Homo sapiens 181 cttcatgtgg caaaatccc 19 182 20 DNA Homo sapiens 182 tgtgctgtca atttggcatc 20 183 20 DNA Homo sapiens 183 aagaagaaat ggggcatagg 20 184 22 DNA Homo sapiens 184 tgtatttgga gacagttccc ac 22 185 19 DNA Homo sapiens 185 aacaatcagt ggcgtggcg 19 186 22 DNA Homo sapiens 186 gacatccagg aggacaggaa ag 22 187 21 DNA Homo sapiens 187 gcagcctctt tcactccata c 21 188 22 DNA Homo sapiens 188 cattgtgtca ggtgatgaaa ag 22 189 20 DNA Homo sapiens 189 ttcatttcta ggcatcgcag 20 190 22 DNA Homo sapiens 190 cattagcagg aggatcaaaa ag 22 191 21 DNA Homo sapiens 191 tctagggcta ttttttggca c 21 192 19 DNA Homo sapiens 192 cgctcccttt caaaatcac 19 193 21 DNA Homo sapiens 193 tgcgagactt tgatgagaca c 21 194 20 DNA Homo sapiens 194 agaccatcag ggaggagaac 20 195 18 DNA Homo sapiens 195 tgtgccagca accaaatc 18 196 22 DNA Homo sapiens 196 gctggagatg aagctgaaga ac 22 197 18 DNA Homo sapiens 197 tttccacttc accgaggg 18 198 21 DNA Homo sapiens 198 ccatgttttg tctgttgtgc c 21 199 22 DNA Homo sapiens 199 cacccatcaa cccatcatct ac 22 200 22 DNA Homo sapiens 200 aggcacaaca gacaaaacat gg 22 201 22 DNA Homo sapiens 201 aagcatgatg tagtagtgac cc 22 202 25 DNA Homo sapiens 202 cttgggtagt tttggattca ggtgc 25 203 27 DNA Homo sapiens 203 agatccattg aagacatttg aggagtg 27 204 20 DNA Homo sapiens 204 gattgacata catttgcttc 20 205 20 DNA Homo sapiens 205 tacagtgaag agaaatccag 20 206 18 DNA Homo sapiens 206 tggaattaga catgcaaa 18 207 19 DNA Homo sapiens 207 tgaagaggat aagtcggtc 19 208 18 DNA Homo sapiens 208 tataatcgct gatgctgc 18 209 26 DNA Homo sapiens 209 accaggccag agtcattaaa ctgatc 26 210 25 DNA Homo sapiens 210 ccgaaaagat gcacaaatat agccc 25 211 26 DNA Homo sapiens 211 ctcaaaactt cattctaatt gtgccc 26 212 18 DNA Homo sapiens 212 agataagcgt gcgtcaac 18 213 20 DNA Homo sapiens 213 tcttatggga attgttagca 20 214 19 DNA Homo sapiens 214 ttatgactgg ttcctcctc 19 215 18 DNA Homo sapiens 215 tcatcaacat ttcccagc 18 216 21 DNA Homo sapiens 216 gaaatactgg agatgagtct g 21 217 19 DNA Homo sapiens 217 gagcttaaga gcttccacc 19 

We claim:
 1. An isolated nucleic acid comprising any one of SEQ ID NOs: 1-4 and 9-126, or of a complementary nucleotide sequence.
 2. An isolated nucleic acid comprising at least eight consecutive nucleotides of a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126, or of a complementary nucleotide sequence.
 3. An isolated nucleic acid comprising at least 80% nucleotide identity with a nucleic acid comprising any one of SEQ ID NOs: 1-4 and 9-126, or of a complementary nucleotide sequence.
 4. The isolated nucleic acid according to claim 3, wherein the nucleic acid has 85%, 90%, 95%, or 98% nucleotide identity with the nucleic acid comprising any one of SEQ ID NOs: 1-4 and 9-126, or of a complementary nucleotide sequence.
 5. An isolated nucleic acid that hybridizes under high stringency conditions with a nucleic acid comprising any one of SEQ ID NOs: 1-4 and 9-126 or of a complementary nucleotide sequence.
 6. An isolated nucleic acid comprising a nucleotide sequence as depicted in any one of SEQ ID NOs: 1-4 and 9-126 or of a complementary nucleotide sequence.
 7. A nucleotide probe or primer specific for any one of ABCA5, ABCA6, ABCA9, and ABCA10 genes, wherein the nucleotide probe or primer comprises at least 15 consecutive nucleotides of a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126 or of a complementary nucleotide sequence.
 8. A nucleotide probe or primer specific for an ABCA5 gene, wherein the nucleotide probe or primer comprises a nucleotide sequence of any one of SEQ ID NOS:127-144 or a complementary nucleotide sequence.
 9. A nucleotide probe or primer specific for an ABCA6 gene, wherein the nucleotide probe or primer comprises a nucleotide sequence of any one of SEQ ID NOs: 145-172, or of a complementary nucleotide sequence.
 10. A nucleotide probe or primer specific for an ABCA9 gene, wherein the nucleotide probe or primer comprises a nucleotide sequence of any one of SEQ ID NOs: 173-203, or of a complementary nucleotide sequence.
 11. A nucleotide probe or primer specific for an ABCA10 gene, wherein the nucleotide probe or primer comprises a nucleotide sequence of any one of SEQ ID NOs: 204-217 or of a complementary nucleotide sequence.
 12. A method of amplifying a region of the nucleic acid according to claim 1, wherein the method comprises: a) contacting the nucleic acid with two nucleotide primers, wherein the first nucleotide primer hybridizes at a position 5′ of the region of the nucleic acid, and the second nucleotide primer hybridizes at a position 3′ of the region of the nucleic acid, in the presence of reagents necessary for an amplification reaction; and b) detecting the amplified nucleic acid region.
 13. A method of amplifying a region of the nucleic acid according to claim 12, wherein the two nucleotide primers are selected from the group consisting of a) a nucleotide primer comprising at least 15 consecutive nucleotides of a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126 or of a complementary nucleotide sequence; b) a nucleotide primer according to claim 7; c) a nucleotide primer comprising a nucleotide sequence of any one of SEQ ID NOs: 127-217, or a nucleic acid having a complementary sequence.
 14. A kit for amplifying the nucleic acid according to claim 1, wherein the kit comprises: a) two nucleotide primers whose hybridization position is located respectively 5′ and 3′ of the region of the nucleic acid; and, optionally, b) reagents necessary for an amplification reaction.
 15. The kit according to claim 14, wherein the two nucleotide primers are selected from the group consisting of a) a nucleotide primer comprising at least 15 consecutive nucleotides of a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126, or of a complementary nucleotide sequence; b) nucleotide primer according to claim 7; c) nucleotide primer comprising a nucleotide sequence of any one of SEQ ID NOs: 127-217, or a nucleic acid having a complementary sequence.
 16. The nucleotide probe or primer according to claim 7, wherein the nucleotide probe or primer comprises a marker compound.
 17. A method of detecting a nucleic acid according to claim 1, wherein the method comprises: a) contacting the nucleic acid with a nucleotide probe selected from the group consisting of 1) a nucleotide probe comprising at least 15 consecutive nucleotides of a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126, or of a complementary nucleotide sequence; 2) a nucleotide primer according to claim 7; 3) a nucleotide probe comprising a nucleotide sequence of any one of SEQ ID NOs: 127-217, or of a complementary nucleotide sequence; and b) detecting a complex formed between the nucleic acid and the probe.
 18. The method of detection according to claim 17, wherein the probe is immobilized on a support.
 19. A kit for detecting the nucleic acid according to claim 1, wherein the kit comprises a) a nucleotide probe selected from the group consisting of 1) a nucleotide probe comprising at least 15 consecutive nucleotides of a nucleotide sequence of any one of SEQ ID NOs: 1-4 and 9-126, or of a complementary nucleotide sequence; 2) a nucleotide primer according to claim 7; and 3) a nucleotide probe comprising a nucleotide sequence of any one of SEQ ID NOs: 127-217, or of a complementary nucleotide sequence, and, optionally, b) reagents necessary for a hybridization reaction.
 20. The kit according to claim 19, wherein the probe is immobilized on a support.
 21. A recombinant vector comprising the nucleic acid according claim
 1. 22. The vector according to claim 21, wherein the vector is an adenovirus.
 23. A recombinant host cell comprising the recombinant vector according to claim
 21. 24. A recombinant host cell comprising the nucleic acid according claim
 1. 25. An isolated nucleic acid encoding a polypeptide comprising an amino acid sequence of any one of SEQ ID NOS: 5-8.
 26. A recombinant vector comprising the nucleic acid according to claim
 25. 27. A recombinant host cell comprising the nucleic acid according to claim
 25. 28. A recombinant host cell comprising the recombinant vector according to claim
 26. 29. An isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-8; b) a polypeptide fragment or variant of a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-8; and c) a polypeptide homologous to a polypeptide comprising amino acid sequence of any one of SEQ ID NOS: 5-8.
 30. An antibody directed against the isolated polypeptide according to claim
 29. 31. The antibody according to claim 30, wherein the antibody comprises a detectable compound.
 32. A method of detecting a polypeptide, wherein the method comprises a) contacting the polypeptide with an antibody according to claim 31; and b) detecting an antigen/antibody complex formed between the polypeptide and the antibody.
 33. A diagnostic kit for detecting a polypeptide, wherein the kit comprises a) the antibody according to claim 31; and b) a reagent allowing detection of an antigen/antibody complex formed between the polypeptide and the antibody.
 34. A composition comprising the nucleic acid according to claim 1 and a physiologically-compatible excipient.
 35. A composition comprising the recombinant vector according to claim 21 and a physiologically-compatible excipient.
 36. Use of the nucleic acid according to claim 1 for the manufacture of a medicament intended for the prevention and/or treatment of a subject affected by a dysfunction in the reverse transport of cholesterol.
 37. Use of a recombinant vector according to claim 21 for the manufacture of a medicament for the prevention and/or treatment of subjects affected by a dysfunction in the lipophilic subtance transport.
 38. Use of any one of isolated ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides comprising an amino acid sequence of SEQ ID NOS: 5-8 for the manufacture of a medicament intended for the prevention and/or treatment of subjects affected by a dysfunction in the lipophilic subtance transport.
 39. A composition comprising a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-8, and a physiologically-compatible excipient.
 40. Use of any one of isolated ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides comprising an amino acid sequence of any one of SEQ ID NOs: 5-8 for screening an active ingredient for the prevention or treatment of a disease resulting from a dysfunction in the lipophilic subtance transport.
 41. Use of a recombinant host cell expressing any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides comprising an amino acid sequence of SEQ ID NOs: 5-8 for screening an active ingredient for the prevention or treatment of a disease resulting from a dysfunction in the lipophilic subtance transport.
 42. A method of screening a compound active on cholesterol metabolism, an agonist, or an antagonist of any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides, wherein the method comprises a) preparing a membrane vesicle comprising at least one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides and a lipid substrate comprising a detectable marker; b) incubating the vesicle obtained in step a) with an agonist or antagonist candidate compound; c) qualitatively and/or quantitatively measuring a release of the lipid substrate comprising the detectable marker; and d) comparing the release of the lipid substrate measured in step b) with a measurement of a release of a labeled lipid substrate by a membrane vesicle that has not been previously incubated with the agonist or antagonist candidate compound.
 43. A method of screening a compound active on cholesterol metabolism, an agonist, or an antagonist of any one of ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides, wherein the method comprises a) incubating a cell that expresses at least one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptides with an anion labeled with a detectable marker; b) washing the cell of step a) whereby excess labeled anion that has not penetrated into the cell is removed; c) incubating the cell obtained in step b) with an agonist or antagonist candidate compound for any one of the ABCA5, ABCA6, ABCA9, and ABCA10 polypeptide; d) measuring efflux of the labeled anion from the cell; and e) comparing the efflux of the labeled anion determined in step d) with efflux of a labeled anion measured with a cell that has not been previously incubated with the agonist or antagonist candidate compound.
 44. An implant comprising the recombinant host cell according to claim
 23. 